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Quantitative determination of steviol glycosides

Written by Admin Camag on July 12, 2022. Posted in Case Studies.

This case study demonstrates a rapid characterization of Stevia formulations by a selective derivatization of steviol glycosides and additionally, it shows the concept of confirming identity of the different steviol glycosides by coupling with mass spectrometry.

Introduction

HPTLC is a powerful technique with regard to high sample throughput capacity, short analysis time, and low running costs. Our case study demonstrates a rapid characterization of Stevia formulations by a selective derivatization of steviol glycosides and additionally, it shows the concept of confirming identity of the different steviol glycosides by coupling with mass spectrometry.

Challenge

Often chromatographic techniques require a time and cost intensive sample preparation step. In many cases HPTLC allows the analysis of samples after a minimal sample preparation (e.g. dissolving) in combination with the separation of several samples side by side. Quantification of the separated compounds is possible by densitometry using CAMAG’s TLC Scanner. In addition, the identity can be proven by eluting the spot of interest using CAMAG’s TLC-MS Interface and coupling it to any mass spectrometer.

Description of the procedure and documentation

Sample and standard preparation are performed according to Morlock et al. (Journal of Chromatography A, 1350 (2014) 102-111 and CBS 109, p.10-12). All parameters (conditions for sample application, chromatogram development, and evaluation) are logged into the visionCATS software, which controls all major CAMAG instruments needed for the HPTLC analysis. The samples and standards are applied band-wise on the HPTLC plate using the ATS 4, which guarantees precise and reliable application. Then the HPTLC plate is developed using the ADC 2. For the analysis of steviol glycosides the developed plate is dipped into β-naphtol reagent, heated at 122°C for 5 minutes, and documented by the TLC Visualizer under white light and UV 366 nm. For quantification each track is scanned in absorption mode at 500 nm with the TLC Scanner and evaluated with visionCATS software. If further investigations of separated sample components are of interest, their eluted zones can be analyzed with other techniques like MS, NIR, and NMR (off-line hyphenation).

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Images of the results: densitogramm, calibration curve and image comparison of Stevia formulation samples, standards and leaves after derivatization
02

HPTLC-MS full scan mass spectrum of Rebaudioside A zone showing most pronounced signals at m/z 989.6 [M+Na]⁺

Benefits of using modern HPTLC for the analysis of food samples

HPTLC visualizes at the first glance the similarities and differences between samples and references.
By using internal or external chemical reference standards compounds can be quantified precisely with the TLC Scanner.
HPTLC is also used for assays (potency), determination of purity (adulteration/fraud), and stability studies (shelf life).
The entire sample is detectable on the plate and allows evaluation of all components even if some of them remain at the application zone or in the solvent front.
HPTLC can analyze samples with high matrix content because of the disposable stationary phase.
HPTLC can be used as high throughput and low-cost technique for the analysis of food samples.
Due to the non-destructive nature of the chromatographic method, analytes can be eluted after separation from the plates using CAMAG’s TLC-MS Interface and analyzed by hyphenated techniques (e.g. MS, NIR, NMR).

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Quantification of methylglyoxal in Manuka honey – A simple HPTLC based approach

Written by Admin Camag on April 15, 2022. Posted in CBS Articles.

Honey is one of the most frequently tested natural food products. In recent years, Manuka honey has gained popularity because of its high antibacterial activity [1]. Methylglyoxal (MGO) was identified as one of the major contributors to its antibacterial property. MGO is present in high concentrations in manuka honey and is directly responsible for its potency. This makes the Manuka honey exclusive and high-priced as compared to the other traditional kinds of honey. Manuka honey from New Zealand usually contains 40 to 800 mg/kg of MGO but can even contain up to 1900 mg/kg [2]. To avoid adulteration of Manuka honey products, a strict quality regulation regarding its origin, purity, and quality needs to be fulfilled and is a prerequisite for the UMF™ (Unique Manuka Factor) grading [2]. It mostly reflects the MGO amount in the honey but also considers other authenticity markers.

In the following application, we focus on the MGO quantification using HPTLC with subsequent substance confirmation by MS measurement. The high viscosity and high sugar content of honey make it a very complex and matrix-rich sample to analyze. HPTLC is a convenient, fast, and efficient separation technique that enables the development of analytical methods without the need for complicated sample preparations or high investments [3]. Low cost and short analysis time per sample are given by the parallel analysis of many samples on one plate. Furthermore, the high matrix tolerance of HPTLC offers additional opportunities to existing routine methods.

Six different commercially available Manuka honey samples were analyzed. MGO shows a mesomeric effect and reacts immediately with water to form either methylglyoxal monohydrate or methylglyoxal dihydrate [4]. Only a small amount of around 1% MGO remains unreacted. Direct detection of MGO in Manuka honey is difficult with conventional methods. In this application, MGO is converted to the stable 2-methylquinoxaline by derivatization with 1,2-phenylenediamine [5]. The stable form is then used as the reference. For confirmation of the method and determination of the recovery rate, regular honey samples were spiked with MGO and 1,2-phenylenediamine. Other derivatization options were tested but the reaction with 1,2-phenylenediamine performed best. Water and honey matrix were tested, to confirm that the optimized reaction conditions provide reproducible results for both matrices.

Standard solutions

The standards are prepared by dissolving 100 μL of Sigma-Aldrich ~40% aqueous MGO solution (exact content known from batch specification) in 20.0 mL of water. 800 μL of the stock solution is further diluted with water to 10.0 mL and 0.2% (2 mg/mL) of the reactant Supelco 1,2-phenylenediamine powder is added. All standard solutions are stored at 8°C for two days before use to achieve reproducible reaction of MGO with 1,2-phenylendiamine. Longer storage time (>3 days) leads to partial degradation of 2-methylquinoxaline.

Sample preparation

Honey samples are prepared at 100–150 mg/mL (in the shown examples: honey sample solutions of 100 mg/mL in case of sample numbers 1, 3, 5, and 150 mg/mL in case of honey samples 2, 4 and 6 were applied in a higher volume due to the expected lower amount of MGO). To each sample 0.2% of 1,2-phenylenediamine is added, e.g., sample 1, 4.0 g honey diluted in 40 mL solution of water /ethanol in 3:2. To the solution, 0.2% (2 mg/mL) of the reactant 1,2-phenylenediamine is added. Before using the samples, they need to be stored at 8°C for two days to complete the reaction.

Chromatogram layer

Supelco HPTLC silica gel 60 F₂₅₄ 20 x 10 cm are used. The plates are pre-washed with the developing solvent (up to 70 mm) and dried before use.

Sample application

Between 0.3–9.0 μL of the samples and standard solutions are applied as areas 5 x 3 mm [2] with the ATS 4 (y = 10 mm, x = 15 mm). This step is necessary because of the high matrix and high application volumes of the honey samples.

Chromatography

After drying, the plate is developed in a Twin Trough Chamber (20 x 10 cm, both troughs filled, 15 minutes waiting time before use, without filter paper) with ethyl acetate – acetonitrile 85:15 (V/V) to the migration distance of 50 mm (from the lower edge), followed by drying for 5 min at 60°C with a plate heater.

Post-chromatographic derivatization

After development, the plate is dried and then derivatized by dipping in an anisaldehyde-sulfuric acid reagent (0.5 mL p-anisaldehyde, 85 mL methanol, 10 mL glacial acetic acid, 5 mL sulfuric acid 98%). Blue zones of 2-methylquinoxaline (product of the reaction ofMGOwith 1,2-phenylenediamine) appear at R_F 0.8. The yellow zone at R_F 0.7 is unreacted 1,2-phenylendiamine.

Documentation

Images of the plate are captured with the TLC Visualizer in white light.

Densitometry

Absorbance measurement at 480 nm is performed with CAMAG TLC Scanner 3 (slit dimension: 2.00 x 0.20 mm, scanning speed: 20 mm/s, data resolution: 100 μm/step).

Mass spectrometry

The target zones are marked in UV light 254 nm. Afterwards, they are directly eluted from the plate with acetonitrile – water – formic acid 95:5:0.1 (V/V) and an MS-Interface, and measured with a single quadrupole MS.

Results and discussion

As demonstrated, MGO can be identified and quantified in different honey samples within the concentration range of 50 to 600 mg/kg. The conversion of MGO into the more stable compound 2-methylquinoxaline allows for an easy evaluation of theMGO content. A recovery study was performed using regular honey to simulate the honey matrix. It was spiked with a known amount of MGO standard solution, followed by the addition of 1,2-phenylenediamine. The measured (and calculated) MGO amount allowed for the correlation of the actual amount of MGO in the Manuka honey samples. The recovery study showed a detectable MGO amount of around 90%. The correlated MGO amount in Manuka samples was calculated accordingly. One of the samples (sample 1) showed a lower MGO content than indicated by the supplier. This might be because of the degradation of the MGO during storage. Samples 2 and 6 only showed MGO concentrations of 50 and 100 mg/kg. These Manuka honey samples are considered of lower quality; although no MGO concentration was provided by the supplier.

In summary, the analysis of MGO in a complex and challenging food matrix like honey was described. The target analyte could be easily separated and detected without time-consuming and laborintensive sample preparation. The flexible set-up enabled a combination with MS measurements. Screening and method development capabilities were shown by the application of 27 tracks on one plate. The study clearly differentiated various honey qualities (referring toMGO content) on the market. Though the analysis of MGO is challenging, MGO content could be well quantified in the expected range. A fast, cheap, and simple quantification of MGO can be accomplished with HPTLC.

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Mass spectra of 2-methylquinoxaline (m/z = 145.0 [M+H⁺] and 186.0 [M+ACN+H⁺]) and 1,2-phenylendiamin (m/z = 150.0 [M+ACN+H⁺])
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Reaction mechanism of MGO and 1,2-phenylenediamine
03

HPTLC chromatograms in white light after derivatization with anisaldehyde (left) and the corresponding densitogram (right) measured at 480 nm (zones at R_F 0.80)
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Calibration curve for quantification of MGO in Manuka honey samples

Literature

[1] E. Mavric, et al. (2008) Mol Nutr Food Res
[2] https://newzealandhoneyco.com/pages/mgo-vs-umfcalculator- manuka-honey
[3] M. Schulz et al. (2009) Analytix Reporter, Issue 5
[4] https://magritek.com/2021/01/29/identification-andquantification- of-methylglyoxal-in-manuka-honey/
[5] E. Dimitrova (2013), Master thesis, TU University of Graz, Austria
[6] Merck (unpublished results)

Further information on request from the authors.

Contact: Dr. Monika Bäumle, Global Product Manager TLC, Merck, monika.baeumle[at]merckgroup.com.

The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.

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Identification and quantification of glucose degradation products

Written by Admin Camag on April 15, 2022. Posted in CBS Articles.

The subject of this article was investigated by Prof. Dr. Martin Brandl, Anette Engels, Dr. Matthias Vogel, Dr. Thomas Zapf and Sarah Leitzen [1]. Prof. Dr. Martin Brandl is a pharmacist and professor at the Department of Physics, Chemistry and Pharmacy at the University of Southern Denmark (SDU), Odense, Denmark and supervisor of Sarah Leitzen’s doctoral thesis. Anette Engels, Dr. Matthias Vogel and Dr. Thomas Zapf work at the Federal Institute for Drugs and Medical Devices (BfArM) in Bonn. Sarah Leitzen is a pharmacist and PhD student working at both BfArM and SDU.

Introduction

Sterile glucose solutions are commonly used as reconstitution solvents or diluents for injectable drugs and also for peritoneal dialysis solutions. In Germany, regulatory requirements for the different strengths of glucose solutions used for parenteral administration are regulated and published as standard marketing authorizations. During heat sterilization of glucose solutions for parenteral use, a variety of glucose degradation products (GDPs) may be formed. GDPs can cause cytotoxic effects after parenteral administration of these solutions. Therefore, the aim of the current study was to develop a simple and quick HPTLC method by which the major GDPs can be identified and (summarily) quantified in glucose solutions for parenteral administration. All GDPs were derivatized with o-phenylenediamine (OPD). The identity of the resulting GDP derivatives (quinoxalines) during method validation was confirmed via mass spectrometry

Standard solutions and pre-chromatographic derivatization

For each degradation product (glyoxal (GO), methylglyoxal (MGO) , glucosone (2-KDG) , 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and the impurity 5-hydroxymethylfurfural (5- HMF)), individual 0.5 mg/mL solutions are prepared with fresh ultrapure water, as well as a mix of all seven GDPs. For quantification, calibration standards containing all seven GDPs at concentration levels of 1–75 μg/mL are prepared. All solutions also contain 50 mg/mL glucose and 0.75 mg/mL OPD. They are left to stand in the dark for 16 hours at room temperature.

Sample preparation and pre-chromatographic derivatization

An artificial mix is prepared to simulate expected concentrations of GDPs in an autoclaved 5% glucose solution containing GO and MGO (1.0 mg/mL, each), 2-KDG (7.0 mg/mL), 3-DG (45.0 mg/mL), 3-DGal (25.0 mg/mL), 3,4-DGE and 5-HMF (5.0 mg/mL, each). The mix also contains glucose in a concentration of 50 mg/mL and OPD as derivatization reagent in a concentration of 0.75 mg/mL. Samples are analyzed after a 16 h reaction time at room temperature.

Chromatogram layer

HPTLC glass plates silica gel 60 F₂₅₄ (Merck), 20 x 20 cm are used.

Sample application

Samples and standard solutions are applied as bands with the Automatic TLC Sampler (ATS 4), band length 10.0 mm, distance between tracks of 19.0 mm, distance from lower edge 29.0 mm. 10 μL for sample and standard solutions are applied.

Chromatography

Plates are developed in the Twin Trough Chamber (TTC 20 x 20 cm) with chamber saturation (with filter paper) for 20 min, development with 25 mL of 1,4-dioxane – toluene – glacial acetic acid 49:49:2 (V/V) (each trough) to the migration distance of 160 mm (from the lower edge), followed by drying for 10 min.

Editor’s Note: in this special case, a migration distance of 160 mm leads to a significantly improved separation on HPTLC with the selected solvent system. Usually, a migration exceeding 80 mm on HPTLC plates does not improve resolution due to increasing diffusion

Post-chromatographic derivatization (second derivatization)

The plates are sprayed with the Derivatizer (yellow nozzle, level 4, 2 mL) with thymolsulfuric acid reagent (2 mL of a solution of 0.5 g of thymol in a mixture of 5 mL of sulfuric acid and 95 mL of ethanol 96%) and heated at 130˚C for 10 minutes on the TLC Plate Heater.

Authors’ Note: While establishing the method, two identical plates were run simultaneously (in separate twin trough chambers with chamber saturation), where one of the two plates was not treated with thymol-sulfuric acid reagent for substance confirmation by mass spectrometry.

Documentation

Images of the plate are captured with the TLC Visualizer in UV 254 nm, UV 366 nm, and white light.

Densitometry

The HPTLC plate was scanned in absorbance mode at 330, 370, and 420 nm, and in fluorescencemode at 366>/400 nm.

Mass spectrometry

One of the duplicate plates (plate without 2^nd derivatization step) is used for substance confirmation. The zones are localized with the help of the plate derivatized with thymol-sulfuric acid reagent. Zones of interest are scraped off at the expected position and extracted three times, using 200 μL of methanol each time, and centrifuged for 2 min at 16,000 rpm/21,130 rcf. The supernatants are evaporated with nitrogen and the residues are finally reconstituted in 200 μL of a 5 mM ammonium acetate buffer solution (pH = 3.5). The solutions are analyzed in positive electrospray ionization mode [1].

Editor’s Note: The zones can also directly be eluted to an MS for substance confirmation with the TLC-MS Interface 2. However, this equipment was not available at the BfArM.

Results and discussion

Glucose, as a highly polar molecule, does not react with OPD, or only to a lesser degree, and maintains its hydrophilic character without forming the quinoxaline system. Therefore, glucose is observed at the application position.

The presented method was successfully validated using the ICH Q2(R1) guideline. For 2-KDG, the linearity of the method was demonstrated in the range of 1–50 μg/mL, for 5-HMF and 3,4-DGE 1–75 μg/mL, for GO/MGO 2–150 μg/mL, and for 3- DG/3-DGal 10–150 μg/mL. All GDPs achieved a limit of detection (LOD) of 2 μg/mL or less and a limit of quantification (LOQ) of 10 μg/mL or less. R² was 0.982 for 3.4-DGE, 0.997 for 5-HMF, and 0.999 for 2-KDG, 3-DG/3-DGal, and GO/MGO. The intraday precision was between 0.4 and 14.2% and the accuracy, reported as%recovery, between 86.4 and 112.7%. The proposed HPTLC method appears to be an inexpensive, fast, and sufficiently sensitive approach for routine quantitative analysis of GDPs in heat-sterilized glucose solutions.

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Graphical abstract from [1] (https://creativecommons.org/licenses/by/4.0/legalcode).
02

Chromatograms of the derivatized seven GDPs and a mixture (= spiked glucose solution) in the solvent 1,4-dioxane – toluene – glacial acetic acid 49:49:2 (V/V). The chromatograms are shown in UV 366 nm without (left) and with (right) subsequent 2^nd derivatization with thymolsulfuric acid reagent at concentration levels of 0.5 mg/mL [1]. (https://creativecommons.org/licenses/by/4.0/legalcode).
03

Overview of the obtained R_F values and signal responses of each analyte at different wavelengths *without and **with second derivatization.
04

HPTLC chromatograms in UV 366 nm after both derivatizations (ROI until R_F 0.6). Track 1 shows the spiked 5% glucose solution spiked GDP-Mix-OPD); tracks 2–8 the calibration solutions in ascending concentrations (1.0 μg/mL–100.0 μg/mL GDPs) [1]. All solutions contain 50 mg/mL glucose. (https://creativecommons.org/licenses/by/4.0/legalcode).
05

Calibration curve of GO/MGO at concentrations of 2–150 μg/mL (measured in absorbance mode at 330 nm) [1]. (https://creativecommons.org/licenses/by/4.0/legalcode).

Literature

[1] Leitzen et al. PLoS ONE (2021), https://doi.org/10.1371/journal.pone.0253811

Further information on request from the authors.

Contact: Sarah Leitzen, Federal Institute for Drugs and Medical Devices, Bonn, Germany, sarah.leitzen[at]bfarm.de

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HPTLC detection of falsification in drugs

Written by Admin Camag on April 15, 2022. Posted in CBS Articles.

Mrs. Carole Vrignaud and her colleagues at the Anti-counterfeiting Laboratory (LCAC) at Sanofi Tours, France, employ chromatographic separation techniques, especially HPTLC, to develop new qualitative analytical methods for detection of falsified drugs, in a variety of sample matrices. The world drug market is about 1000 billion € (2019). The World Health Organization (WHO) considers that half of the drugs sold through the internet are fakes. This is why Sanofi group in 2008 created this unique laboratory to fight against drug falsification using various analytical methods, including HPTLC. The main objective is to confirm the presence of active ingredients (API) or excipients (preservatives, flavoring…) in each Sanofi formulation. HPTLC is also often used to detect degradation of products stored under inappropriate conditions, as well as unexpected compounds, allowing characterization of the falsified products. To detect falsification of drugs, a generic HPTLC method was developed.

HPTLC is well suited for a rapid parallel screening of many samples. Up to 10 samples can be analyzed in less than one hour. The developed method is simple and can be perform ed with little consumption of solvents and reagents.

Standard solutions

20 mg of API no. 1, 55 mg of API no. 2, 53 mg of API no. 3, and 51 mg of impurity of API no. 2 are individually dissolved in 10 mL of water (each).

Sample preparation

One tablet of each suspected case of falsification is milled, and then extracted with 7.5 mL of water by vortexing. After centrifugation, the filtered supernatant is transferred into a 5 mL volumetric flask and filled up to the mark.

Chromatogram layer

HPTLC plates silica gel 60 F₂₅₄ (Merck), 20 x 10 cm, pre-washed with methanol, are used.

Sample application

Samples and standard solutions are applied as bands with the Automatic TLC Sampler (ATS 4), 14 tracks, band length 8.0 mm, distance from left edge 20.0 mm, distance from lower edge 8.0 mm. 1.0 μL of sample solutions and 3.0 μL of standard solutions are applied.

Chromatography

In the ADC 2 with chamber saturation (20 min) and after conditioning at 33% relative humidity for 10 min using a saturated solution of magnesium chloride, development with dichloromethane – ethanol – water – formic acid 9:9:1.5:0.25 (V/V) to the migration distance of 50 mm (from the lower edge), followed by drying for 5 min.

Post-chromatographic derivatization

First, the plates are manually sprayed with Dragendorff’s reagent (commercial solution) and dried for 60 s at room temperature. Second, the plates are manually sprayed with sodium nitrite solution (5%) and dried for 30 s at room temperature.

Documentation

Images of the plates are captured with the TLC Visualizer in UV 254 nm before derivatization and in white light after each derivatization step.

Densitometry

Absorbance measurement is performed with the TLC Scanner 3 and visionCATS at 272 nm, slit dimension 5.0 mm x 0.2 mm, measurement speed 20 mm/s, spectra recording from 200 to 450 nm.

Results and discussion

A representative plate with real samples is shown. During method development simulated and spiked samples gave the same R_F values as the standards and were well separated from matrix components. Any positive identification of API no. 2 and no. 3 can be confirmed by spectral comparison of samples and standards as well as the expected presence of impurity of API no. 2. Detection of API no. 1 (cases no. 1–7) is achieved after derivatization in white light. Results are shown for selected drug products as well as the identification of three active ingredients expected in different oral formulations. HPTLC is also used to estimate each quantity. API no. 3 is not present in the composition of the original tablets (cases no. 8–9). However, some previous cases were identified as counterfeit tablets containing API no. 3 instead of API no. 2.

In the shown real cases no falsification was detected. Nevertheless, the approach has proved suitable by several positive cases. HPTLC is therefore used at Sanofi as high throughput screening technique.

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HPTLC chromatogram (top) in UV 254 nm prior to and (bottom) in white light after derivatization; tracks 1&10: API no. 1; tracks 2–8: cases no. 1–7; track 9: API no. 3; track 11: API no. 2; track 12: impurity of API no. 2; tracks 13–14: cases no. 8–9
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(Left) UV spectrum API no. 3 (red) and samples (cases no. 1-7); (right) UV spectrum API no. 2 (red) and samples (cases no. 8–9)

Further information on request from the authors.

Contact: Carole Vrignaud, 30-36 avenue Gustave Eiffel, 37100 Tours, France, carole.vrignaud[at]sanofi.com

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Parameters of High-Performance Thin-Layer Chromatography (Part 1)

Written by Admin Camag on April 14, 2022. Posted in White Paper.

TLC is a simple, quick, and flexible way to analyze samples, and there is no right or wrong way of doing it. However, if efficient, standardized, and reproducible results are desired, HPTLC is the right choice. HPTLC implies the control of all parameters of the process.

Sample Preparation

Sample preparation is at the beginning of the HPTLC process and can significantly influence the result. For practical reasons, it should be kept as simple as possible. Three main steps are included:

The sample homogenization, comprising drying, grinding, freezing, lyophilizing and/or milling step(s);
The choice of solvents;
The extraction method.

Choosing a suitable extraction solvent involves several considerations:

It should be non-hazardous (health, explosion, flammability), stable, and not reactive with the sample;
It should be inexpensive and easily removable from the plate.

Polar solvents are water, ethanol, and methanol, medium polarity solvents are ethyl acetate, dichloromethane, and ethers, non-polar solvents are toluene, and cyclohexane. Some extraction methods are:

Maceration and percolation;
Reflux/Soxhlet;
Steam distillation (for essential oils / terpenes);
Supercritical fluid extraction;
Shaking;
Extraction in the ultrasonic bath (sonication);
Warm water-bath (boiling);
Liquid-liquid extraction;
Microwave-assisted extraction;
Accelerated solvent extraction.

For samples with unknown properties, or as a convenient starting point, CAMAG proposed a simple extraction scheme covering different solvent polarities: 0.5 g of the powdered sample (amount can be adjusted) are extracted by sonication for 10 minutes with 5.0 mL of the following solvents:

Neat solvents and their mixtures	Acidic mixtures	Basic mixtures
Heptane/Toluene	Methanol – acetic acid 9:1 (V/V)	Methanol – ammonia 25% 8:2 (V/V)
Butyl methyl ether	Water – acetic acid 9:1 (V/V)	Water – ammonia 25% 8:2 (V/V)
Dichloromethane
Ethyl acetate
Acetone
Acetonitrile
Ethanol
Methanol
Water
Ethanol – water 7:3 (V/V)
Methanol – water 8:2 (V/V)

The 15 test solutions can be analyzed in parallel with a mobile phase from the literature or a universal solvent system.

Application

During sample application, the following basic requirements must be met. The position of the sample must be precise to ensure a simultaneous start of the chromatography for all samples on the plate. This is prerequisite for the comparability of R_F values of separated zones. For that, a standardized plate layout is adopted. Precise application volumes are required for qualitative and quantitative analysis. A common problem is sample overloading, which decreases the separation power and causes tailing of zones. Furthermore, it is also clear that damages to the layer during sample application can affect the movement of the mobile phase.

Contact or Spray-on Application

During contact application, the sample solution is transferred onto the plate with a (fixed volume) capillary or a syringe. The volume and volatility of the solvent affect the size of the applied spot. The strength of the solvent determines the distribution of the sample components in the applied spot (circular chromatography during application). Compact spots improve the chromatography resolution (see example below, where a test dye solution dissolved either in methanol or in toluene was applied by contact application, Figure 1A).

Contact application as spots (A) and spray-on application as narrow bands (B)

Spray-on application vaporizes the sample solution with a gas stream as it is dispensed from a syringe. Before it reaches the layer, a significant part of the solution’s solvent is evaporated, and mainly the sample is transferred to the plate. That way, sharp lines are applied for improved separation during chromatography, regardless of the strength of the solvent (in Figure 1B). The spray-on technique can concentrate large sample volumes into narrow bands.

Band or Spot Application?

During visual evaluation, the resolution seems to improve with the length of the separated zones, but that is just an optical illusion. Nevertheless, evaluation of the homogenous center part of a band improves the signal to noise ratio during the generation of peak profiles.

Most pharmacopoeias prefer band application in HPTLC due to better suitability for visual evaluation.

Comparison of 2 mm and 8 mm band length

Plate Layout

The standard format of the HPTLC plate is 20 x 10 cm. A precise application position is essential for proper identification of separated zones. For the application of samples with respect to plate layout, certain parameters (see Figure 3) need to be considered.

Plate layout proposed by the HPTLC Association and adopted by The United States Pharmacopoeia (USP), European Pharmacopoeia (Ph. Eur.), and British Pharmacopoeia (BP).

INSTRUMENTATION

CAMAG® HPTLC PRO Module APPLICATION

The degree of automation and productivity are key factors for the HPTLC laboratory. The Module APPLICATION is part of the CAMAG® HPTLC PRO SYSTEM, a fully automated sample analysis and evaluation system using HPTLC plates (20 x 10 cm), which is best suited for routine quality control of analytes extracted from complex matrices and provides reproducible and reliable results.

The Module APPLICATION plays a crucial role within the HPTLC PRO SYSTEM as it largely determines the quantification accuracy. Designed for highly precise application of samples as bands onto HPTLC glass plates, it supports the autonomous application of up to 75 samples on up to five HPTLC plates. To avoid cross contamination, the Module APPLICATION employs an S/N marked syringe generating a highly effective separation bubble between the rinsing solvent and the sample solution. To ensure highly precise application of samples as narrow bands, the Module APPLICATION laser-controls the needle distance and dosage speed, which vary depending on the sample solvent used.

The HPTLC PRO SYSTEM and the individual modules are controlled by visionCATS, a state-of-the-art software platform for workflow organization and data evaluation management.

CAMAG® HPTLC PRO Module APPLICATION

The ATS 4 is a highly advanced, versatile and powerful system, enabling the fully automated application of 66 samples from vials or 96 samples from well plates using either the spray-on technique or spot application by contact transfer. Any x- and y- position on the TLC plate can be selected for application. Samples can also be applied as rectangles, a feature that is very useful for large volumes of samples containing the analyte in very low concentration. Prior to chromatography, the rectangles are focused to narrow bands with a solvent of high strength. Designed for routine use and high sample throughput, the automated ATS 4 is suited for qualitative and quantitative analyses as well as for preparative separations, while offering great flexibility by supporting any plate format up to 20 x 20 cm. The ATS 4 is controlled by visionCATS HPTLC Software.

CAMAG® Automatic TLC Sampler 4 (ATS 4)

CAMAG® Linomat 5

A widely used sample applicator is the semi-automatic Linomat 5, a device that introduced all advantages of the spray-on technique to HPTLC. Precise volume dosage and exact positioning combined with flexibility and convenient handling are among the features of the instrument. The user only loads the sample manually into a syringe and selects the y-position of the application; the instrument manages all other parameters of the application process. Like every other software-controlled HPTLC instrument from CAMAG, the Linomat 5 is controlled by visionCATS HPTLC Software.

CAMAG® Linomat 5

Development

Development is one of the core elements of HPTLC. During development, the analyst needs to be aware of several parameters, which will affect the chromatography and the reproducibility of the results. The following paragraphs will discuss the influence of the separation distance and humidity in the chromatography, and the different chamber configurations.

Separation Distance

In (HP)TLC, the velocity of the mobile phase depends on capillary forces and is not constant as in HPLC or GC. With increasing separation distance, the velocity of the mobile phase decreases because of increasing resistance caused by the silica gel layer, which affects resolution. With a decreasing velocity of the mobile phase diffusion increases. That is the reason why zones get broader in the upper part of the chromatogram. Even though their distance still increases, resolution/ separation of zones does not improve beyond 6 cm separation distance (see Figure 7). This is why many compendia adopted 6 cm as optimum separation distance.

Figure 7: Separation of chamomile oil on HPTLC silica gel 60 plates. Mobile phase: toluene – ethyl acetate 19:1 (V/V), derivatization by dipping in 10% sulfuric acid in methanol

Separation of chamomile oil on HPTLC silica gel 60 plates. Mobile phase: toluene – ethyl acetate 19:1 (V/V), derivatization by dipping in 10% sulfuric acid in methanol

Influence of the Relative Humidity

Silica gel has a hydrophilic nature. Silanol groups on its surface interact strongly with water vapor adsorbed from the environment. This leads to deactivation of the layer, reducing the analyte’s interaction with the stationary phase and increasing R_F values. Exposing silica gel to high temperatures (e.g. 120°C) or low relative humidity removes adsorbed water (activation). The silanol groups become more active, and the R_F values of the analytes will be lower. Because HPTLC is performed in an open system, results are always affected by the relative humidity of the environment. To ensure reproducible results, it is recommended to adjust the activity of the plate prior to development by exposing the plate for a certain time to a defined relative humidity established over a saturated salt solution.

Saturated salt solution	% relative humidity
NaCl	75
NaBr	58
KSCN	47
MgCl₂	33
CaCl₂	31
KCOOH	20
(Molecular sieve)	0–5

HPTLC Chamber Configurations

Chamber configuration and the corresponding gas phase will affect the chromatographic result. In standardized HPTLC, a saturated Twin Trough Chamber (TTC) is generally utilized, but also othermodes of operation are possible:

Unsaturated TTC: The development starts by introducing the plate immediately after the developing solvent is poured into one of the troughs of the TTC. The other trough remains empty. During development, components of the mobile phase may evaporate from the plate. At the same time, the developing solvent is evaporating into the chamber. Equilibrium between liquid and gas phase (saturation) is not achieved; the chamber remains “unsaturated”.
Saturated TTC: Both troughs of the TTC are charged with developing solvent. The rear trough is fitted with filter paper (saturation pad). With the lid closed (e.g. for 20 minutes), equilibrium of the developing solvent and its vapor in the chamber is achieved before the plate is introduced for development. Unless the developing solvent is a neat solvent, the component(s) with the higher vapor pressure will have a higher proportion in the gas phase.
Pre-conditioning in TTC: The developing or another conditioning solvent is poured into the rear trough of the TTC, which may contain a filter paper (saturation pad), and the chamber is saturated (e.g. for 20 minutes) with the lid closed. Then, the plate is inserted into the front trough and, thus, exposed to the gas phase. After a certain time (e.g. 10 minutes) of pre-conditioning, the developing solvent is introduced into the front trough, and chromatography starts.

Special features of the unsaturated TTC are:

Sharper zones;
Higher R_F values;
Sometimes better separation of critical pairs;
Longer developing time;
Chromatography susceptible to variation in temperature, humidity and chamber geometry;
Results less reproducible.

Special features of saturated TTC and pre-conditioning are:

Broader zones;
Lower R_F values;
Shorter developing time;
Chromatography is less affected by variation in temperature, humidity and chamber geometry;
Reproducible results;
Pre-conditioning further decreases the R_F values.

Practical tip: The level of developing solvent must always be below the application position on the plate. In HPTLC, the level is 5 mm with an application position of 8 mm.

Post-chromatographic Drying of the Plate

Once the mobile phase reached the targeted solvent front position, the plate should be immediately removed from the chamber and dried to stop chromatography and suppress any diffusion of separated components. During the drying, the plate is kept in a vertical position.

INSTRUMENTATION

CAMAG® HPTLC PRO Module DEVELOPMENT

Designed to develop 20 x 10 cm HPTLC glass plates, the Module DEVELOPMENT obtains highly reproducible analytical results and offers high flexibility regarding the choice of the solvents and configuration of the separation process. Its unique chamber geometry allows for the full control of the gas phase prior to and during chromatography. Prior to development, the stationary phase can be pre-dried, activated, and pre-conditioned. During development, the gas phase can be actively circulated to achieve the best possible separation of the analytes. After development, the mobile phase is drained off the chamber and a running blower removes the residual mobile phase from the stationary phase. Combined with the Modules PLATE STORAGE and APPLICATION, the Module DEVELOPMENT allows for the sequential execution of several analysis files, enabling the autonomous development of up to five different HPTLC glass plates with up to three different developing solvents.

CAMAG® HPTLC PRO Module DEVELOPMENT

CAMAG® Automatic Developing Chamber ADC 2

The Automatic Developing Chamber 2 (ADC 2) is an instrument for reproducible development, performing the development step fully automated, and independent of environmental effects. The activity and pre-conditioning of the layer, chamber saturation, developing distance and final drying can be preset and automatically monitored by the ADC 2. The fully automated ADC 2 employs a conventional 20 x 10 cm Twin Trough Chamber for development, allowing to retain the chromatographic conditions of already existing analytical procedures while excluding environmental and operational effects. The option Humidity Control allows reproducible chromatography at defined activity of the layer. This feature is essential in method development when the influence of relative humidity shall be investigated.

CAMAG® Automatic Developing Chamber ADC 2

CAMAG® AMD 2 System Automated Multiple Development

The separation of complex samples is a challenging task for every chromatographic system, particularly when the sample components span a wide polarity range. The software-controlled AMD 2 is designed to solve difficult separation problems that cannot be handled by isocratic HPTLC. The AMD procedure offers an excellent solution as it allows stepwise gradient elution over increasing separation distances. As a result, acids, bases, neutral, hydrophilic, and lipophilic substances can be separated in a single AMD run. This makes AMD suitable for a variety of applications. The technique is frequently used in lipid analysis and in routine analysis of drinking water. Pigment formulations with a complex composition, resins as well as additives of mineral oil products are other typical applications of AMD analysis.

CAMAG® AMD 2 System Automated Multiple Development

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Screening for chlorinated paraffins in vegetable oils and dietary supplements by pSPE

Written by Admin Camag on April 4, 2022. Posted in CBS Articles.

Introduction

Chlorinated paraffins (CP) are complex mixtures of polychlorinated n-alkanes with 10 to 30 carbon atoms and variable chlorine contents (30–70%). CP are categorized by their carbon chain length as short- (SCCP; C₁₀–C₁₃), medium- (MCCP; C₁₄–C₁₇), and long-chain (LCCP; C₁₈–C₃₀) CP and they are widely used for technical applications, e.g., they are added to plasticizers, paints, and flame retardants. CP are known to be persistent, and their bioaccumulation depends on the carbon chain length and the degree of chlorination. Because of their high toxicity, SCCP were classified as persistent organic pollutants (POP) according to the Stockholm Convention in 2017. MCCP and LCCP have similar chemical properties as SCCP, however, there have been only few studies about their toxicity and bioaccumulation, and their metabolism and distribution in the environment up to now, why they also pose a very high risk to humans. Due to their chemical structure, the degradation of CP in the environment is negligible, while accumulation in lipophilic tissues is very likely and high quantities might occur in lipids. CP were already detected in vegetable oils, milk, and dairy products, as well as in human milk fat and additionally in food supplements with high fat content. The analysis of CP is a very challenging task because CP are complex mixtures of polychlorinated n-alkanes, comprising thousands of congeners. The separation of these complex mixtures of unknown isomers and congeners is even not possible by gas chromatography (GC). Therefore, the presented screening method for CP uses the pSPE concept that quantifies CP as the sum. Based on the cost-effective and efficient HPTLC technique, pSPE guarantees reliable results for many samples simultaneously in a short time [1].

The determination of CP by applying the pSPE concept was successfully developed for a rapid and selective screening of CP as the sum. After sulfuric acid treatment and liquid-liquid partition into n-hexane, pSPE was performed on silica gel plates employing a twofold development. The analytes, which were focused in a single target zone, were determined by a densitometric absorption scan after derivatization with o-tolidine showing the total CP content, and the amounts were calculated as the sum by means of a reference CP. A comparison of the results obtained for the total CP in dietary supplements by pSPE with visual detection (pSPE–VIS) and by GC with highresolution mass spectrometry (GC–HRMS) proved the pSPE approach as reliable tool for screening purposes.

Standard solutions

For method development, technical CP mixtures (SCCP with chlorine contents of 51.5%, 55.5%, and 63% and MCCP with chlorine contents of 42%, 52%, and 57%) are diluted in n-hexane to a concentration of 25 ng/μL. For the determination of the limit of decision and quantitation, recovery experiments in vegetable oils, and the analysis of dietary supplements, the reference CP (MCCP with a chlorine content of 52%) is diluted to concentrations of 0.075–0.8 ng/μL, 0.75–10 ng/μL, and 0.25–10 ng/μL, respectively. The internal standard (ISTD) 4,4-DDT is prepared at a concentration of 400 μg/mL (stock solution) and used at a final concentration of 6 ng/μL in all samples and standards. [1] For GC–HRMS measurements, SCCP calibration standards with chlorine contents of 51.5%, 53.5%, 55.5%, 59.25%, and 63% and MCCP standards with chlorine contents of 42%, 47%, 52%, 54.5%, and 57% are prepared by respective dilutions of technical CP mixtures to a concentration of 10 ng/μL. The ISTD α-PDHCH and ε-HCH are added at concentrations of 0.075 and 0.05 ng/μL to all samples and standards. [1]

Sample preparation

Vegetable oil is treated according to Coelhan [2] with somemodifications. After the addition of 15 μL of the ISTD 4,4-DDT stock solution to 250 mg of vegetable oil, 500 μL of n-hexane and 2.5 mL of concentrated sulfuric acid are added and the mixture is shaken for 3 min at 2200 rpm prior to centrifugation for 30 min. The n-hexane phase is separated, and the extraction is repeated with 500 μL of n-hexane under the same conditions. Both n-hexane extracts are combined and used for pSPE [1]. Oil-based dietary supplement capsules (250 mg of oil) are extracted by the same procedure as described above for the vegetable oil (following the procedure of the sulfuric acid treatment of the sample solution in n-hexane) including the addition of 7.5 μL of the ISTD α-PDHCH prior to the extraction (for GC–HRMS analysis). Furthermore, the combined n-hexane extracts (≈1 mL) are shaken with 2 mL of sulfuric acid for 4 min prior to centrifugation for 5 min. After separation of the n-hexane phase, the sulfuric acid treatment is repeated under the same conditions. A 300 μL aliquot of the n-hexane extract is used for pSPE and a 400 μL aliquot of the n-hexane extract including 2 μL of the ISTD ε-HCH is used for GC–HRMS. [1]

Chromatogram layer

HPTLC plates silica gel 60 (Merck), 20 x 10 cm are used.

Sample application

30 μL of samples and standards are applied as areas of 6 mm x 3 mm with the Automatic TLC Sampler (ATS 4), 22 tracks, distance from the left edge 13.0 mm and from the lower edge 8.0 mm. After application, plates are dried in a fume hood for 5 min.

Chromatography

Plates are developed twofold in the ADC 2 after activation at 33% relative humidity for 5 min using a saturated solution of magnesium chloride. The 1^st development is performed with cyclohexane – toluene 94:6 (V/V) to a migration distance of 80 mm and the 2^nd development is done with dichloromethane – n-hexane 90:10 (V/V) to a migration distance of 50 mm followed by a drying time of 3 min after each development.

Post-chromatographic derivatization

The plate is immersed into a solution of o-tolidine (4% in acetone) using the Chromatogram Immersion Device with an immersion speed of 3 cm/s and an immersion time of 1 s, dried for 4 min in a stream of cold air, and irradiated with UV-C light inside a self-made irradiation device using a cycle with irradiation and cooling steps [1].

Documentation

Images of the plate are documented with the TLC Visualizer in white light.

Densitometry

Absorbance measurement at 645 nm is performed with the TLC Scanner 4 with a scanning speed of 20 mm/s, a data resolution of 100 μm/step, and a slit dimension of 5 mm x 0.30 mm using the manual detector mode applying a quick scan range of 47–53 mm on the track of the most concentrated standard. For quantitation, the respective peak areas are used.

Gas chromatography coupled to high-resolution mass spectrometry

GC–HRMS measurements with electron capture negative ionization (ECNI) are performed according to the method of Krätschmer et al. [3] and quantitation is done according to Sprengel et al. [4] and Reth et al. [5].

Results and discussion

The determination of CP as the sum was performed by the straightforward pSPE approach. The target analytes were selectively separated from the matrix and additionally focused in a single and sharp target zone since all representatives of the chemical group of CP have almost identical properties in regard to a chromatographic separation. Best results were obtained by a twofold development on HPTLC silica gel with cyclohexane – toluene 94:6 (V/V) for the 1^st development and dichloromethane – n-hexane 90:10 (V/V) for the 2^nd development. In the target zone, the selective determination of the total CP as the sum was possible directly on the plate after derivatization with o-tolidine and UV irradiation by means of an absorption scan at 645 nm. For quantitation, a reference CP (MCCP with a chlorine content of 52%) was used.

01

Plate image after pSPE and derivatization with o-tolidine in white light of (1–4) from left to right, SCCP (chlorine content 55.5% and 63%), MCCP (chlorine content 52% and 57%), 225 ng CP/zone; (5–9) different vegetable oils, 7.5 mg sample/zone; (10) a reagent blank.
02

Corresponding 3D densitogram of the absorption scan at 645 nm

Limits of decision and quantitation for the reference CP were determined according to the DIN 32645 calibration method [6] in the range of 2.25–24 ng CP/zone (corresponding to 0.3–3.2 mg CP/kg of vegetable oil) and 1.5–4.9 ng CP/zone (corresponding to 0.2–0.7 mg CP/kg of vegetable oil), respectively, with relative standard deviations (RSD) <5%. Thus, the presented pSPE–VIS approach delivers a suitable screening tool for the analysis of the total CP down to ~1 mg/kg of vegetable oil, while for lower limits, sample application volume can be increased.

Recoveries for CP in sunflower oil, olive oil, and rice bran oil at levels of 5, 10, and 30 mg reference CP/kg were very close to 100% after the subtraction of a slight impurity originating from the sample preparation. With precision of recovery expressed as RSD <4%, the method was very well repeatable for all tested vegetable oils and spiking levels.

01

Mean recoveries of CP from vegetable oils in % ± standard deviation (n = 4) at spiking levels of 5, 10, and 30 mg reference CP/kg of oil. Modified from [1].
02

Total CP amounts in six dietary supplement samples from the European market by pSPE–VIS and GC/ECNI–HRMS in mg reference CP/kg of sample ± standard deviation (n = 4). Modified from [1].

Comparison of results obtained by pSPE–VIS and GC–HRMS for six dietary supplement samples (oil-based food supplements) showed generally conformity. The total CP contents determined by pSPE–VIS were in the same order of magnitude compared to the contents analyzed by GC–HRMS and results were well repeatable. Only one sample (sample 3) showed a clearly higher content by pSPE, however, displayed a different color of the target zone that evidently originated from comigrated matrix components. In general, large deviations in results are well known for the very challenging CP analysis, which depend on the used GC–MS set-up including the calibration standards and the calibration method.

In summary, the pSPE screening approach for the determination of the total CP provides a very useful alternative to the time-consuming and very complicated GC approaches that analyze the individual components and calculate the sum afterwards. Furthermore, in terms of a rapid screening, pSPE offers the chance to save time and costs, because only the suspected and questionable samples need additionally to be analyzed by GC/ECNI–HRMS.

Literature

[1] C. Oellig et al., J Chromatogr A (2019) 460380.
[2] M. Coelhan, Anal Chem (1999) 4498.
[3] K. Krätschmer et al., J Chromatogr A (2018) 53.
[4] J. Sprengel et al., Rapid Commun Mass Spectrom (2018) 49.
[5] M. Reth et al., J Chromatogr A (2005) 225.
[6] Deutsches Institut für Normung, DIN 32645, Beuth, Berlin, 2008.

Further information on request from the authors.

Contact: PD Dr. habil. Claudia Oellig, Department of Food Chemistry and Analytical Chemistry (170a), Institute of Food Chemistry, University of Hohenheim, 70599 Stuttgart, claudia.oellig[at]uni-hohenheim.de

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The HPTLC Association Substance Database: a useful tool for identification of compounds.

Written by Admin Camag on March 31, 2022. Posted in CBS Articles.

The idea of developing a substance database for natural products arose from a previous collaboration on HPTLC of flavonoids and phenolic acids, between CAMAG and the ZHAW(Zurich University of Applied Sciences, Wädenswil). The project investigated the chromatographic behavior of about 72 substances. In a pilot phase in collaboration with the company Extrasynthese we worked on two smaller projects on iridoids and coumarins to practically implement the database concept.

Introduction

The HPTLC Association Substance Database is a systematic collection of data, easily accessible electronically from the website of the HPTLC Association [1]. It contains retention and spectral data of representative substances from different classes, analyzed with different developing solvents and derivatization reagents.

With the substance database, HPTLC laboratories have a free and convenient tool to help with identification of zones in a chromatogram. The user can compare R_F values, and colors prior to and after derivatization, and UV spectra of unknown zones with those of the references. As the collection will be regularly expanded with substances not limited to constituents of herbal drugs, different fields of application can benefit from the HPTLC Association substance database. Iridoids and coumarins are described in this article.

Standard solutions

Iridoids and coumarins were dissolved in methanol at a concentration of 1.0 mg/mL, and concentrations were adjusted if needed.

Chromatogram layer

HPTLC plates silica gel 60 F₂₅₄ (Merck), 20 x 10 cm are used.

Sample application

2.0 μL of solutions were applied as bands with the Automatic TLC Sampler (ATS 4), 15 tracks, band length 8.0 mm, distance from left edge 20.0 mm, distance from lower edge 8.0 mm.

Chromatography

Plates were developed to 70 mm (from the lower edge) in the ADC 2 with chamber saturation (20 min, with filter paper) and after activation at 33% relative humidity for 10 min using a saturated aqueous solution of magnesium chloride. Different mobile phases were investigated (see table), followed by drying for 5 min.

Post-chromatographic derivatization

Iridoids were derivatized with anisaldehyde sulfuric acid (AS) and vanillin sulfuric acid (VS) reagents. Coumarins were derivatized with AS, potassium hydroxide (KOH), and natural product (NP) / polyethylene glycol (PEG) reagents. AS reagent: slowly and carefully mix 170.0 mL of ice-cooled methanol with 20.0 mL of acetic acid and 10.0 mL of sulfuric acid; allow the mixture to cool to room temperature, then add 1.0 mL of anisaldehyde (p-methoxy benzaldehyde); VS: dissolve 1 g of vanillin in 100 mL of ethanol 96% and carefully add 2 mL of concentrated sulfuric acid; KOH: dissolve 5 g potassium hydroxide in 100 mL of methanol (96%); NP: dissolve 1 g of 2-aminoethyl diphenylborinate in 200 mL of ethyl acetate; PEG: mix 10 g of polyethylene glycol 400 (macrogol) with 200 mL of dichloromethane.

Documentation

Images of the plates were captured with the TLC Visualizer 2 in UV 254 nm, UV 366 nm and white light, and in UV 366 nm and white light after derivatization.

Densitometry

Absorbance measurements at 254 nm with the deuterium lamp for iridoids, and at 313 nm with the mercury lamp for coumarins were performed with TLC Scanner 4 and visionCATS, slit dimension 5.00 mm x 0.20 mm, scanning speed 20 mm/s. Spectra of the corresponding zones were recorded in the absorbance-reflectance mode from λ=190–450 nm using a combination of deuterium and tungsten lamps with a slit size of 5.00 mm x 0.20 mm.

Results and discussion

Analysts who want to identify unknown zones in the HPTLC chromatogram are limited to the availability of reference standards in their laboratory. To help with selecting appropriate standards, the substance database can be a useful screening tool. For each class of compounds, six steps were taken: evaluation of the existing HPTLC methods, selection of three developing solvents of diverse properties, evaluation of the derivatization reagents, analysis of all substances of each class, and creation of the documentation.

The HPTLC Association provides method documents (PDF format) for each class and all evaluated developing solvents for a convenient check of available substances.

The documentation of the database also includes PDF files for each individual substance including peak profiles from image, UV spectra and images of chromatograms prior to and after derivatization.

Users of visionCATS can download all the data as comparison files and import their own data for comparison.

01

Heptane is not included in selectivity groups. AS: anisaldehyde sulfuric acid. VS: vanillin sulfuric acid, KOH: potassium hydroxide; NP: natural product; PEG: polyethylene glycol.
02

Method document for iridoids, developing solvent A
03

Substance document of agnuside, developing solvent A
04

Use of the comparison file from the substance database for establishing the presence of harpagoside and harpagide in Harpagophytum procumbens.

Literature

[1] T. K. T. Do, R. De Vaumas, E. Reich. J Planar Chromatogr 1638 (2021). https://doi.org/10.1016/j.chroma.2020.461830

Further information on request from the authors.

Contact: Dr. Tiên Do, CAMAG Laboratory, Sonnenmattstrasse 11, 4132 Muttenz, Switzerland, tien.do[at]camag.com

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Principles of Planar Chromatography

Written by Admin Camag on January 13, 2022. Posted in White Paper.

Planar Chromatography

Planar chromatography is a form of liquid chromatography: A liquid mobile phase dissolves the substances of a mixture and transports them through a flat (planar) stationary phase. Different affinities of substances to the two phases afford separation as they cause different retardation with respect to the velocity of the mobile phase. In column chromatography, this affects the time of elution (retention time) while in planar chromatography the position of substance relative to the front of the mobile phase on the plate is changed. Because planar chromatography takes place in a developing chamber, also a gas phase is established, which can influence the separation. There are two forms of planar chromatography: Paper Chromatography and Thin-Layer Chromatography (TLC). Planar chromatography is performed in off-line mode, which means that the principle steps of the process (sample application, development, derivatization, detection, evaluation) are independent in time and location. This leads to some specific advantages:

great flexibility in the choice of stationary and mobile phases
parallel analysis and visual comparison of multiple samples and standards
single-use of the stationary phase reduces the need for sample clean-up
possibility of multiple detections without the need to repeat chromatography
convenient chemical derivatization of separated substances and detection of biological activity
low cost
rapid

The Thin-Layer Chromatography (TLC) System

The TLC system comprises the mobile phase, stationary phase, and gas phase. The first two phases interact directly with the sample components and achieve their separation. The gas phase is established in the developing chamber and affects the dry part of the stationary phase.

Figure 1: The TLC system

The Stationary Phase

Different adsorbents can be used as stationary phase in TLC but silica gel (in normal phase mode) is the most common one. Due to its porous (6 nm) structure, silica gel has a large surface featuring siloxane and silanol groups. These can interact with different functional groups of the sample components based on polarity, acidity and non-specific interaction. Silica gel is a strong adsorbent and interacts with water from the lab atmosphere. This process affects the activity and the selectivity of the stationary phase.

Figure 2: Stationary phases (based on silica gel)

Chemical modification of the silanol groups leads to bonded phases, of non-polar (reversed-phase – RP) or polar (normal phase – NP) nature and quite different selectivity.

Name (Merck)	Functionality	Polarity	Principal use as
RP 2 (C2)	Dimethyl	Non-polar	RP (partition)
RP 8 (C8)	Octyl	Non-polar	RP (partition)
RP 18 (C18)	Octadecyl	Non-polar	RP (partition)
Silica gel (SiOH)	Silanol	Polar	NP (adsorption/partition)
Amino (NH2)	3-aminopropyl	Middle polar	RP/NP (partition/adsorption)
Cyano (CN)	3-cyanopropyl	Middle polar	RP/NP (partition/adsorption)
Diol (OH)	Spacer bonded propanediol	Middle polar	NP (adsorption)

For non-polar reversed-phases, polarity decreases and hydrophobicity increases with the chain length (C2 > C8 > C18). The dominant retention mechanism is partition: the analytes are distributed between the bonded (liquid) phase and the mobile phase. Plates labelled “wettable” (W) are compatible with higher amounts of water in the mobile phase.

The dominant retention mechanism on normal phases is adsorption: the analytes and the molecules of the mobile phase compete for adsorption on the stationary phase.

Practical tip: on silica gel, the R_F values decrease with the number and increasing polarity of the functional groups: hydrocarbons (low polarity) usually migrate to a higher R_F with a medium- polarity mobile phase. In contrast, acids, alcohols or sugars (higher polarity) tend to stay closer to or at the application position with a medium-polarity mobile phase. On a reversed-phase, the longer the analyte’s carbon chain, the lower its R_F values (higher retention).

Figure 3: The dominant retention mechanisms for normal phases (adsorption) and non-polar reversed-phases (partition).

To create a stable adsorbent layer on the support, most commercial plates contain an organic polymeric binder. Some plates have a so-called fluorescence indicator that is excited at a specific wavelength, e.g. short wave UV (254 nm), labeled as F₂₅₄.

F	fluorescence indicator
254	excitation wavelength for F
PSC/PF	preparative layer chromatography, thickness > 0.25 mm
RP 2, 8, 18	reversed-phase with chain length/C-atoms 2, 8, 18
W	water resistant / wettable layer (most common for RP-18 plates)
s	acid stable indicator
60	average pore diameter in Angstrom (60 Å = 6 nm)
AMD	automated multiple development, thickness ~ 0.1 mm
MS	mass spectrometry, thickness ~ 0.1 mm
G	gypsum (used as binder)

The Mobile Phase

The mobile phase is the solvent or mixture of solvents that transports the analytes through the stationary phase and contributes to the selectivity of the chromatographic systems.

Note: mobile phase and developing solvent are not the same thing. The mobile phase is the liquid that migrates through the stationary phase. Its composition (proportion of solvents) may change during chromatography due to evaporation and other factors. The developing solvent has a defined composition and is filled into the developing chamber.

The solvent strength refers to the ability of the solvent to elute the solutes from the stationary phase. By increasing the mobile phase strength, the retention of the solute decreases and the R_F value increases. In adsorption chromatography, solvent strength is commonly called “polarity”, while in reversed-phase, it can be expressed as lipophilicity. A classification of solvent strength (Polarity index P’) for normal phase separation is shown in the table below.

Solvent	Polarity index P’	Selectivity Group
Hexane	0.0	–
Heptane	0.1	–
Isopropyl ether	2.4	I
Diethyl ether	2.8	I
2-propanol	3.9	II
n-butanol	3.9	II
1-propanol	4.0	II
Ethanol	4.3	II
Methanol	5.1	II
Tetrahydrofuran	4.0	III
Acetic acid	6.0	IV
Dichloromethane	3.1	V
Ethyl acetate	4.4	VI
Methylethyl ketone	4.7	VI
Dioxane	4.8	VI
Acetone	5.1	VI
Acetonitrile	5.8	VI
Toluene	2.4	VII
Benzene	2.7	VII
p-xylene	2.5	VII
Nitromethane	6.0	VII
Chloroform	4.1	VIII
Water	10.2	VIII

Figure 4: Illustration of the effect of increasing mobile phase strength on the RF values of a dye mixture on silica gel. The more ethyl acetate (P’ of 4.4) is in the mobile phase, the higher the RF of the zones.

In reversed phase chromatography, the solvent strength increases with the increasing amount of organic modifier and decreasing amount of water.

The mobile phase selectivity describes the interaction of the solvent with different sample molecules. R. L. Snyder proposed eight solvent selectivity groups with different properties (Figure 6). Solvents from different selectivity groups can have the same polarity (e.g. tetrahydrofuran and 1-propanol). This can affect the migration order of analytes in a sample.

Figure 5: Eight solvent selectivity groups with different properties

In the practical example shown in Figure 6, a test dye mixture was analyzed with four different developing solvents of the same strength on silica gel. The solvents in cases (a) and (b), ethyl acetate and acetone, belong to group VI, causing very similar migration orders, except for the purple zone. By changing to a solvent from group II (1-propanol), the yellow and blue zones switch positions, and the gray zone has a lower R_F value (case c). In the last case (d), with neat chloroform (group VIII), nearly all zones change positions.

Figure 6: Analysis of test dye mixture with four different mobile phases of the same strength on silica gel.

The Gas Phase

A feature that distinguishes TLC from other chromatographic techniques is the presence of an additional gas phase. It is formed in the chromatographic chamber by evaporation of solvents. The gas phase interacts with the stationary and mobile phases, before and during chromatography. It is possible to standardize the conditions of the gas phase in order to achieve reproducible results.

In a “classic TLC chamber”, i.e. a twin-trough chamber (TTC) with specific dimensions, containing filter paper, a developing solvent, a stationary phase with samples, and which is closed with a lid, the gas phase is involved in four important processes:

Figure 7: The gas phase is involved in four important processes: saturation (A), pre-conditioning (B), evaporation (C), and secondary fronts (D).

Saturation (Figure 7A): The components of the developing solvent evaporate and eventually establish phase equilibrium with their vapor (e.g. after 20 minutes, when using a filter paper to increase the surface for evaporation). The vapor pressure of the individual components of the developing solvent affects the composition of the gas phase.
Pre-conditioning (Figure 7B): The components of the gas phase are adsorbed on the dry stationary phase, forming a thin layer of solvent molecules. A polar solvent in the gas phase will interact more with the silica gel than one of lower polarity.
Evaporation of the mobile phase during chromatography (Figure 7C): During the development step, a part of the mobile phase may evaporate from the wet stationary phase, leading to increased developing time, particularly if the chamber has not been saturated.
Secondary fronts (Figure 7D): During the development step, an active stationary phase can separate the components of the mobile phase, resulting in the formation of secondary fronts that may influence the separation of the analytes. This effect is weaker with chamber saturation and pre-conditioning of the stationary phase.

In TLC, separation usually takes place in a partial equilibrium between stationary, mobile, and gas phases.

The Retardation Factor

The retardation factor (R_F) in planar chromatography, also known as the retention factor, is a term used to describe the relative positions of zones in the chromatogram. It is calculated based on the ratio of the distances traveled by the center of a zone “A” (b) and distance traveled by the mobile phase front (a) with respect to the application position.

By definition, R_F values are always less than one, and they are displayed with two decimals. IUPAC defines hR_F as R_F multiplied by 100, but most pharmacopoeias and publications use R_F.

Figure 8: Calculation of the retardation factor (R_F)

TLC vs. HPTLC

Thin-layer chromatography (TLC) is a special case of planar chromatography. TLC has been used since the mid-20th century for diverse separation purposes, and until now, it is generally done either manually or with a simple instrumental setup. It is a simple, flexible, and rapid tool for qualitative investigations. However, as there is no agreement on a standardized TLC methodology, it is challenging to get reproducible results between laboratories (if that is the method’s aim).

The term High-Performance Thin-Layer Chromatography (HPTLC) was introduced when plates with smaller particle sizes and dimensions were brought to the market. HPTLC is considered the evolution of classical TLC. With time, HPTLC was clearly defined by compendia and became a concept that prioritizes the quality and reproducibility of the results. The HPTLC concept includes:
- HPTLC glass plates of fixed dimensions (20 x 10 cm) with layers of silica gel (or other adsorbents) with particle sizes between 2 and 10 μm and a fluorescence indicator;
- Simple to sophisticated instruments, available for all chromatography steps;
- HPTLC software;
- Well-defined methods with optimized and standardized parameters;
- Qualified data through method specific system suitability test (SST);
- Validated methods, compliant with cGxP;
- Traceable digital data.
A standardized and optimized TLC method on a 20 x 20 cm plate can be scaled down to run on a 20 x 10 cm HPTLC plate. Thus, HPTLC can be considered as miniaturized TLC.

HOW IT WORKS

SMOOTH & PRECISE OPERATION

01

HPTLC glass plate (20 x 10 cm)
02

TLC plate (20 x 20 cm)
03

Respective particle size

The main differences between TLC and HPTLC are shown in the table below:

	TLC	HPTLC
Principle	Planar chromatography	Planar chromatography
Primary focus	Simplicity, low cost	Reproducibility, separation power
Process	Flexible, no rules	Highly standardized methodology/optimized parameters
Methods	Only a few parameters defined	Well defined and validated
Flexibility	Very high	None for validated methods, high between methods
Target	Rapid, preliminary results	Reliable analytical results
Data structure	Simple chromatograms/photographs	Traceable digital images/scan data qualified by system suitability test (SST) on each plate, cGMP compliant reporting
Samples/references	Side by side on the plate	On the same or on different plates
Plate	TLC any format	HPTLC 20 x 10 cm
Instrumentation	None to simple	Simple to sophisticated
Cost	Very low	Medium to high compared to TLC, low per sample compared to HPLC

Further information on request from the authors.

Contact: Amélie Havard, Daniel Dron, Oril Industrie (Servier), Industrial Research Centre, Department of Analytical Innovative Technologies R&D, 13 rue Auguste Desgenétais, CS 60125, 76210 Bolbec, France amelie.havard@servier.com, daniel.dron@servier.com

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High-throughput analysis of aroma precursors in cocoa and coffee

Written by Admin Camag on September 30, 2021. Posted in CBS Articles.

Dr. Vincent Lebot is a plant breeder and geneticist at CIRAD (Centre International de Coopération en Recherche Agronomique, France) and employs chromatographic separation techniques, especially instrumental planar chromatography, to select genetically improved tropical plant hybrids and varieties of suitable chemotypes. HPTLC is his preferred technique because of its low solvent consumption and running costs and its support of a high-sample throughput.

Introduction

It is estimated that there are around 24,000 varieties of cocoa and more than 30,000 varieties of coffee in the world [1, 2]. Only a few varieties have been evaluated for their quality aiming at specialty and aromatic products, which is an important trait for securing markets, especially for smallholders in small producing countries. During the fermentation and drying processes, aroma precursors present in the green beans are transformed into flavour compounds determining the quality of commercial beans. The chemical analysis of non-volatile compounds is, therefore, an essential step for the selection of varieties with desired quality traits. Alkaloids (theobromine and caffeine) and polyphenols (catechins, proanthocyanidins and anthocyanins) are contributing to the cocoa flavour. Caffeine, trigonelline and chlorogenic acids are known to produce astringent and sour taste and bitterness in coffee but very few varieties have been compared in controlled conditions. Sucrose is also an aroma precursor in coffee and depends on the variety. The objectives of this study were: i) to develop a protocol for the quantification of non-volatile compounds in cocoa and coffee, ii) to analyze and compare cocoa and coffee varieties from diverse geographical origins cultivated in controlled conditions and harvested at full maturity.

Standard solutions

Standard stock solutions of theobromine, trigonelline, caffeine, ideain-3-O-galactoside, cyanidin- 3-O-arabinoside, (–)-epicatechin, (–)-catechin, chlorogenic acid (CGA), neochlorogenic acid (NCGA or 5-O-caffeoylquinic acid), 3,4-, 3,5-, 4,5- dicaffeoylquinic acids, and sucrose are prepared at 1.0 mg/mL with methanol. Standard solutions are prepared at different concentration levels with methanol and stored at 4 °C.

Sample preparation

Cocoa pods and coffee berries are handpicked and selectively harvested when they are completely mature to guarantee the complete uniformity of the material from the different accessions. All samples are collected from a single healthy tree per accession basis. Green beans of cocoa and coffee were then oven dried at 60 °C until constant weight (11% humidity) and milled. For each accession sample, 10 g of powder are mixed in centrifuge tubes with 30.0 mL of acetone for cocoa and 30.0 mL of methanol – water 7:3 (v/v) for coffee, sonicated for 10 min and then centrifuged at 1,585 x g for 10 min. The supernatant is transferred to a vial stored at 4 °C in the dark until analysis.

Chromatogram layer

HPTLC plates silica gel 60 F₂₅₄ (Merck) 20 x 10 cm are used.

Sample application

1.0 μL of standard and sample solutions are applied as bands with the Automatic TLC Sampler (ATS 4), band length 8.0 mm, distance from the left edge 15.0 mm, track distance 8.9 mm.

Chromatography

Plates are developed in the Automatic Developing Chamber (ADC 2) with ethyl acetate – toluene – formic acid – water 7:1:1:1 (v/v) without chamber saturation for cocoa extracts and with ethyl acetate – dichloromethane – formic acid – acetic acid – water 23:6:2:2:2 (v/v) with chamber saturation (10 min, with filter paper) for coffee extracts, to a migration distance of 70.0 mm. For sucrose, plates are developed with acetonitrile – water 87:13 (v/v) without chamber saturation to a migration distance of 85 mm.

Documentation

Images of the plates are captured with the TLC Visualizer in UV 254 nm and UV 366 nm and in white light after derivatization.

Densitometry

Absorbance measurement at 275 nm and 330 nm prior to derivatization and for sucrose at 520 nm after derivatization with TLC Scanner 4 and winCATS, slit dimension 8.00 mm x 0.20 mm, scanning speed 50 mm/s.

Editor’s Note: A slit length of 5.00 mm is usually used for a band length of 8.00 mm.

Derivatization

The plate is immersed into anisaldehyde reagent (for proanthocyanidins) or aniline-diphenylaminephosphoric acid reagent (for sucrose) with the Chromatogram Immersion Device and heated on the TLC Plate Heater at 105 °C for 10 min.

Results and discussion

For repeatability accuracy assessment, linear ranges were computed using the least squares method. Repeatability was confirmed by applying four repetitions of each standard at five different concentration levels (0.1, 0.2, 0.3, 0.4, 0.5 μg/μL) and the variance among repetitions was expressed as the repeatability standard deviation (%RSD) [3]. Peak area measurements were compared to individual standards and corresponding values were quantified in mg/g dry weight [3]. The repeatability of the HPTLC measurements was assessed for each individual standard and the calibration plots were linear for all analytical standards with all R² > 0.99 (P = 0.01). For each compound, %RSD values were low (< 3.5%) indicating that the HPTLC measurements are accurate enough to be used for quantification.

Multivariate analysis of non-volatile compounds (via peak areas obtained by scanning densitometry) in 137 cocoa varieties clearly discriminates the different groups of varieties (Amenolado, Criollo, Forastero, Trinitario). Theobromine was the most important compound in all accessions analyzed, followed by caffeine, epicatechin/catechin, cyanidin, and ideain. Proanthocyanidin B2 was also important and four other proanthocyanidins were minor compounds [3]. The 108 varieties of coffee analyzed were clearly differentiated based on their caffeine content which is significantly correlated with chlorogenic acids. Arabica varieties present low caffeine. Sucrose, trigonelline, caffeine, and eight chlorogenic acids were detected and quantified based on the R_F values of the standards and by matching their UV spectra with those of the samples [3]. Principal component analysis (PCA) of the coffee varieties shows that 73.2% of the total variation is explained by axis 1 and 2. C. arabica are characterized by low alkaloids and CGAs contents and are mostly located on the left side of axis 1, while C. canephora characterized with high alkaloids and CGAs contents are located on the right side of axis 1.C. arabica with < 11 mg/g of caffeine are on the far left end of axis 2 and C. canephora with > 18 mg/g of caffeine are on the far right end of axis 2. Further results and details are available at [3].

01

HPTLC chromatograms of different cocoa varieties (20 tracks, top: UV 254 nm prior to derivatization, middle: white light prior to derivatization, bottom: white light after derivatization with anisaldehyde reagent for polyphenols: (–)-epicatechin: EC and (–)-catechin: GC, and proanthocyanidins: PA, A2, C1, B1, B2 [3]
02

HPTLC chromatograms of different Arabica coffee varieties (20 tracks, top: UV 254 nm prior to derivatization, middle: UV 366 nm prior to derivatization, bottom: white light after derivatization with ADPA reagent.
03

Densitogram of a coffee sample measured at 330 nm prior to derivatization (with increasing R_F values chlorogenic acid (CGA), neo-chlorogenic acid (NCGA or 5-O-caffeoylquinic acid), 4,5-dicaffeoylquinic acid (4,5-di), 3,4-dicaffeoylquinic acid (3,4-di), 3,5-dicaffeoylquinic acid (3,5-di), and three unknown caffeoylquinic acids labelled as CQA (1), CQA (2), CQA (3).
04

Overlay of UV spectra of CGA standard and samples (recorded from 200–550 nm) to determine the optimum wavelength for scanning densitometry (λ_max at 330 nm)
05

PCA of 108 accessions of Coffee arabica (A), C. arabusta (B), C. canephora (C), and hybrids of C. canephora and C. congensis (H). The Arabica varieties (A) are differentiated from Canephora (robusta) varieties by their low chlorogenic acids contents (left of axis 1).

Conclusion

HPTLC is a cost-efficient technique when applied to both qualitative and quantitative assessment of chemical constituents in green cocoa and coffee beans. Compared to other chromatographic techniques, HPTLC presents the outcome as an image of the separated non-volatile compounds on a single plate, detected by UV light. This visible outcome and the simplicity of the technique could allow breeders to run the chromatographic procedure described in this study on hundreds or thousands of varieties and breeding lines. The analysis time for a plate with 20 tracks is comparatively short (approximately 38–42 minutes) and diverse genotypes can be analyzed side by side on the plate, making HPTLC the method of choice for rapid chemometric evaluation of cocoa and coffee varieties and detection of exceptional individuals.

Literature

[1] Laliberté, B. et al. (2012) https://agritrop.cirad.fr/568442/1/document_568442.pdf
[2] Bramel, P. et al. (2017) https://worldcoffeeresearch.org/media/documents/Coffee_ Strategy_Low_Res.pdf
[3] Lebot, V. et al. Gen Res Crop Evol (2020) 67: 895–911

Further information on request from the authors.

Contact: Dr. Vincent Lebot, UMR AGAP, CIRAD, P.O. Box 946, Port-Vila, Republic of Vanuatu, lebot[at]vanuatu.com.vu

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Untargeted Fabry-related Globotriaosylceramide (Gb3) biomarkers in plasma

Written by Admin Camag on September 29, 2021. Posted in CBS Articles.

The research group “Nanosensors and Bioanalytical Systems” consists of researchers from the Spanish National Center for Scientific Research (CSIC, at the Instituto de Carboquímica) and the University of Zaragoza. One of the group’s areas of interest is the use of HPTLC-based techniques for analysis of complex mixtures mostly coming from petroleum conversion, biofuels and lipidomics. In addition to the inherent advantages of HPTLC (flexibility, complete sample detection, etc.), its coupling to any MS instrument through an elution-based interface opens up an unexplored range of analytical possibilities [1].

Introduction

Fabry disease is a severe genetic disease of lysosomal deposition produced by malfunction of an enzyme (α-galactosidase A), which results in a gradual accumulation of Gb₃, among other metabolites, in body fluids.

A number of Gb₃-related molecular species in the plasma of Fabry patients were detected at very low concentrations and demonstrated to be biomarkers, using reversed-phase LC coupled to mass spectrometry. HPTLC has generally not been considered as adequate for detailed characterization of a complex sample of lipids. This perception is not correct since currently HPTLC-MS is a methodology to get more information online in less time from complex biological matrices. HPTLC separation reduces mass spectral complexity, which, in turn, allows for a comprehensive examination of samples, even if incompletely resolved separations are obtained.

The objective of this work is to assess whether an HPTLC densitometry-MS approach using the TLC-MS Interface 2 is adequate to determine Fabry biomarkers related to Gb₃ in human plasma.

A separation of a plasma extract into sphingolipid classes, followed by densitometry and MS coupling provides a simple but powerful approach for the detailed structural elucidation of sphingolipids present in human plasma, allowing their recognition by their m/z, and confirmation by their collision induced dissociation MS/MS data, and/or APCI fragmentation.

The TLC-MS Interface 2 is suitable for the analysis of sphingolipids and provides a rapid, precise, and targeted characterization of selected bands on the plate. In our approach we use ESI-MS and APCI-MS. Intensities of ESI(⁺)-MS ions can be related to the concentration of species.

Samples and Standard solutions

Plasma samples from a Fabry’s patient and a healthy control are obtained from the Institute of Health Sciences (Zaragoza, Spain) after approval of the Ethical Committee of Aragon (CEICA, Spain). Informed consent was obtained from the human subjects.

The investigated Gb3 are untargeted species and no standards are available. Solutions of standards of lyso-Gb₃, Gb₃, lactosylceramide (LacCer), glucosylceramide (GlcCer) which represent the main sphingolipid families are prepared at a final concentration of 0.1 μg/μL each in dichloromethane – methanol 1:1 (v/v).

Sample preparation

Neutral sphingolipid extracts are obtained from plasma using a standard sample preparation procedure which involves centrifugation (10 min at 5000 rpm) to remove precipitated protein, followed by alkaline hydrolysis (75 μL of 2M sodium hydroxide, incubated under magnetic stirring for 2 h at 40 °C), and liquid-liquid (H₂O-methanol) extraction. The lower layer containing the neutral sphingolipids is then transferred to a vial and dried under N₂. Samples are reconstituted in 250 μL of dichloromethane – methanol 1:1 (v/v).

Chromatogram layer

Two LiChrospher plates (20 x 10 cm, Merck) are used. They are pre-washed with methanol and kept in a desiccator in N2 atmosphere until use.

Sample application

Sample and standard solutions are applied as 4 mm bands on the corresponding plate by using the Automatic TLC Sampler (ATS 4). Each of the plasma samples and standards are applied in triplicate. The distance between tracks is 10.6 mm, distances from the left and lower plate edges are 10 mm. One or more tracks are left empty, as blanks. Between 25.0–30.0 μL of plasma sample extracts and 0.1–10.0 μL of standard solutions are applied on the same plate.

Chromatography

Development is performed on two twin plates using the Automated Multiple Development (AMD 2) system under the conditions in figure 1.

Post-chromatographic derivatization

One of the twin plates is immersed into 0.2g of orcinol in 100 mL of 10%H₂SO₄ using the Chromatogram Immersion Device (immersion time: 2 s). The plate is heated during 15 min at 100 °C using the TLC Plate Heater 3.

Densitometry

Absorbance measurement is performed with the non-derivatized plate at 190 nm and with the derivatized plate at 550 nm using the TLC Scanner 3.

Mass spectrometry

Selected zones of the non-derivatized plate are eluted with the TLC-MS Interface 2 (oval head) at a flow rate of 0.2 mL/min with methanol into an ion-trap MS with ESI(⁺) ionization. Gb₃ species with m/z between 1000 and 1200 Da are scanned. HPTLC-ESI(⁺)-MS/MS is used for structure identification. Alternatively, APCI(⁺) ionization is used for structure confirmation by an analysis of derived fragments with m/z <1000.

Results and discussion

Identification and semi-quantification of 19 untargeted molecular species of globotriaosyl-ceramides (Gb₃) in extracts from a Fabry’s plasma patient and a healthy control was performed by HPTLC densitometry-MS. The species found were: five isoforms of saturated Gb₃, seven isoforms of methylated Gb₃, and seven species with three unsaturations (that of sphingosine plus two additional unsaturations). Twelve of these species were previously reported as biomarkers of Fabry’s lysosomal disorder using a LC-MS-based method, and the other seven are structurally similar, closely related to them [2].

Saturated Gb₃ isoforms come from the ESI(⁺)-MS analysis of the peak at 27.7 mm of Fabry extract (and 27.9 mm in the case of control). This is the migration distance of the Gb₃ standard. Ions are mostly detected as sodium adducts. The most preponderant isoform is d18:1;C16:0 (m/z 1046.8 [M+Na]⁺). Confirmation of identity of this most abundant ion [C₅₂H₉₇NO₁₈Na]⁺ is done by ESI-MS/MS by loss of a hexose group, giving a product ion at m/z 885.2.

Methylated and three-unsaturated Gb₃ species were both found in HPTLC-UV peaks at 14.9 (Fabry) and 14.2 mm (control). They should have a priori corresponded to preponderant sphingomyelin (SM) species. The obtained HPTLC-ESI(⁺)-MS showed intensities of 10⁵ arbitrary units (a.u.), and high S/N ratio for SM species. However, we found that some Gb₃-related species are present in low concentration and can co-migrate together with the above SM species. Intensities for these Gb₃ species are 10⁴ a.u. (arbitrary units). The zone between m/z 1000-1300 displays ESI ions that matched with either methylated Gb₃-related isoforms as [M+Na]⁺, or Gb₃-related isoforms or analogues with three unsaturations, as [M+H]⁺. The presence of these structures found by ESI-MS was verified by an analysis of lowmolecular ion fragments obtained using HPTLC-APCI(⁺)- MS [2]. This experiment was performed in duplicate and similar spectra were obtained in each case.

Ion intensities are related to concentration of Gb₃ species for several reasons summarized elsewhere [2]. Saturated Gb₃ in Fabry’s plasma were in higher concentration than in control sample in repeated experiments. ESI-MS profiles for methylated and unsaturated Gb₃ species were qualitatively similar for Fabry and control samples although relative distribution of ions is different.

01

AMD 2 gradient for plasma extract separation (final migration distance of 90 mm, DCM = dichloromethane, MeOH = methanol)
02

Fabry’s Gb3-related biomarkers and semi-quantitative ion abundance in Fabry (blue) and control (red) plasma extracts

Conclusion

HPTLC-MS has been proven to be a simple but powerful approach for the detailed structural elucidation of sphingolipids present in human plasma. The potential of HPTLC-MS for lipidomic research in general has been summarized elsewhere [1].

Literature

[1] V.L. Cebolla et al. J Liq Chromatogr & Rel Technol (2021) https://doi.org/10.1080/10826076.2020.1866600
[2] C. Jarne et al. J Chromatogr A 1638 (2021) 461895

Further information on request from the authors.

Contact: Dr. Vicente L. Cebolla, Instituto de Carboquímica, CSIC, Miguel Luesma Castán, 4. 50018 Zaragoza, Spain, vcebolla[at]icb.csic.es

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