Page 9 | CAMAG

St. John’s Wort versus Counterfeit St. John’s Wort: An HPTLC Study

Written by Admin Camag on July 26, 2019. Posted in Scientific Publications.

Abstract

Hypericum perforatum L. is the most commonly used herb for treating depression. Due to the popularity of this botanical, there is a potential for economically driven adulteration of St. John’s wort (SJW) products. The goal of this study was to investigate SJW ingredients suspected to be adulterated based on simple preliminary HPTLC tests. Commercial samples were analyzed by HPTLC following the United States Pharmacopeia (USP) monograph methodology, with additional visualization under white light. A number of these samples presented odd methanolic solution colors and unconventional HPTLC fingerprints, suggesting the presence of other species and/or extraneous polar additives. To achieve identification and separation of the polar additives, a new reversed-phase HPTLC method was developed. The adulterants were identified as synthetic dyes in the amounts of 0.51 to 1.36% by weight. Identities of the dyes were confirmed by scanning densitometry and HPTLC-MS. A modified USP method with additional detection mode permitted the identification of eight SJW samples adulterated with dyes and six others with flavonoid fingerprints different from those specified by USP from a total of 37 samples of dry extracts, finished products, and bulk raw herb. A decision flowchart is proposed to guide the detection of adulteration of SJW in a systematic fashion.

https://www.ncbi.nlm.nih.gov/pubmed/27343017

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Comprehensive HPTLC fingerprinting for the quality control of Angelica gigas root

Written by Admin Camag on July 26, 2019. Posted in Scientific Publications.

Amélie Havard and Daniel Dron work in the R&D department Analytical Innovative Technologies at the Industrial Research Centre at Oril Industrie, in Bolbec, France. The R&D team specializes in purification processes of intermediates and active pharmaceutical ingredients (APIs) for toxicological, galenical or clinical studies. Part of their work is also the isolation of impurities and production of APIs or impurity reference batches. In 2018, the team launched their preparative chromatography service InnoPrepTM, dedicated to small- and large-scaled purifications. APIs, intermediates and impurities are characterized by MS and NMR. A quantitative 1D and 2D NMR method is currently being developed.

Introduction

Efficient purifications by a selective preparative chromatography are widely used in the manufacturing of API batches. The described procedure helps in the understanding of target molecules and related impurities. Impurities, present at very low levels can be isolated at a high purity, which eases identification and finally improves the process. By providing higher purity products in less time, significant financial gain can be achieved.

The required conditions for an efficient purification (ca. 75% use silica gel at Oril Industrie) are determined by TLC. Then, the purification progress is checked by preparative column chromatography via HPTLC. Twenty fractions were analyzable within 1 hour. TLC/HPTLC is the method of choice due to its simplicity, rapidness and the successful scale up from TLC to preparative separations. HPTLC-MS helped to quickly resolve the composition of a mixture.

Sample preparation

Crude product (0.05 g) dissolved in 5 mL ethyl acetate

Chromatogram layer

TLC plate silica gel 60 F₂₅₄ (Merck), 20 x 5 cm HPTLC plate silica gel 60 F_254s (Merck), 20 x 10 cm

Sample application

Automatic TLC Sampler (ATS 4), bandwise application, up to 20 tracks, band length 8.0 mm, sample volumes of 1–15 μL

Chromatography

In the Twin Trough Chamber 20 x 20 cm (TLC) or 20 x 10 cm (HPTLC) with chamber saturation (with filter paper) for 20 min with different solvents to the migration distance of 100 mm for TLC and 50 mm for HPTLC (both from the lower edge), drying in a stream of cold air for 5 min

Documentation

TLC Visualizer at UV 254 nm

Mass spectrometry

Elution of zones with the TLC-MS Interface (oval elution head) at a flow rate of 0.2 mL/min with methanol – water 1:1 into a Q-TOF-MS (Xevo® G2- XS QTof, Waters), operating in the positive ionization mode (m/z 50–1200)

NMR

Elution of zones with the TLC-MS Interface (oval elution head) at a flow rate of 0.2 mL/min with methanol into a vial, followed by evaporation to dryness, dissolution of the residue in deuterated chloroform (with one drop of sodium deuteroxide solution) and 1H NMR recording (400 MHz, Bruker)

Results and discussion

The aim of this study was to obtain a batch with a purity >99% of the isomer Z, containing <1% of isomer E and < 0.15% of other impurities. By RP-HPLC the isomers were not separated satisfyingly, and TLC was selected for method development to separate the two isomers. Ethyl acetate – methylcyclohexane 9:1 was the best option to separate all compounds at reasonable hRF values allowing a fast purification.

Mass spectra were recorded to characterize the different compounds. The same sodium adduct [M+Na]+ and respective dimer [2M+Na]+ were obtained for both Z/E isomeres. For NMR, the crude product solution was concentrated by a factor of 10, applied (15 μL) on the HPTLC plate, separated, and four zones of each target zone were eluted and combined.

The purification of the crude product (2 kg dissolved in toluene) on a 45-cm column (packed at 40 bars with 40 kg silica gel 60, 15–40 μm, Merck) at a flow rate of 10 L/min with ethyl acetate – methylcyclohexane 9:1 led to a productivity of 20 kg per day. The elution process was monitored online by UV 254 nm detection and in parallel offline by HPTLC.

The different fractions of the target isomer Z were collected and the combined fractions analyzed by NMR. The purity was not sufficient, as the NMR spectrum showed several impurities. Thus, the purification was optimized. The new eluent of dichloromethane – ethanol 19:1 together with a crude product load of 0.5 kg also led to a productivity of 20 kg per day. The purity obtained for the Z isomer was 99.8% with a yield of 88%.

TLC is the best method for development and optimization of purification processes using silica gel. It is simple and allows a rapid upscaling to preparative columns. HPTLC is an efficient tool for offline monitoring of the eluted fractions. Up to 20 fractions can be analyzed in parallel and compared in the HPTLC chromatogram at UV 254 nm, achieving a good overview on the purity and amount of the target compound per fraction.

HOW IT WORKS

SMOOTH & PRECISE OPERATION

01

TLC chromatograms at UV 254 nm of the crude product separated with different mobile phases
02

HPTLC chromatograms at UV 254 nm of the crude product (10 g/L, 1 μL versus 100 g/L,15 μL) and mass spectra (left) versus 1H NMR spectra of isomeres (right)
$Online monitoring of the purification process by LC-UV (254 nm, left) versus offline by HPTLC-UV (individual fractions at 254 nm, right)$
03

Online monitoring of the purification process by LC-UV (254 nm, left) versus offline by HPTLC-UV (individual fractions at 254 nm, right)

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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Effect-Directed Analysis of Water Samples

Effect-directed analysis of a water sample

Written by Admin Camag on July 26, 2019. Posted in CBS Articles.

For more than 10 years effect-directed analysis in combination with TLC has been used and advocated at the Laboratory for Operation Control and Research of Zweckverband Landeswasserversorgung in Langenau [1]. It began with the Aliivibrio fischeri inhibition assay for the detection of baseline toxicity. Now three more bioassays (end points: estrogenic, antibiotic and neurotoxic effects) have been applied. The method is used as a fast monitoring tool for investigation of raw and drinking water. Subsequently a method is described, which was developed in cooperation with Dr. Wolfgang Schulz and Dr. Rudi Winzenbacher (same institution) and Prof. Dr. Wolfgang Schwack, University of Hohenheim, Stuttgart.

Introduction

Through increasing influence of humans, the environment more and more gets polluted with anthropogenic trace substances. Many of these substances are not characterized and their effects on humans and the ecosystem are only inadequately clarified. Therefore it is very important to evaluate the eco(toxicological) relevance of this multitude of substances, classifying relevant from less relevant ones. Here, effect-directed analysis (EDA) can make a contribution as a combination of fractionation, bioassay and chemical analysis.

For fractionation of EDA, HPTLC has proven to be a particularly suitable method. In comparison to HPLC, HPTLC is an open separation system, thereby separated substances are solvent-free and the following in-vitro-bioassay can be performed directly. However, in comparison to HPLC, this method is limited due to the lower separation efficiency. To increase the separation efficiency of HPTLC, in this work a two-dimensional (2D) separation strategy with effect-directed detection was developed.

In the presented method, zones with detectable effect are eluted with the TLC-MS interface and the respective eluate (200 μL) is collected into a CBS 121 3 sample vial. The acetylcholinesterase (AChE) inhibition assay for the detection of neurotoxic effects was performed in both separation dimensions. Through this selective procedure, fractions without AChE-inhibiting effects could be excluded after the first separation dimension (1D) and only the effective fractions were transferred for separation in the second dimension.

Standard solutions

For evaluation of the mobile phases, five standards mixed in methanol with a respective concentration of 10 ng/μL were applied. Additionally, a mix with 50 acetylcholinesterase (AChE)-inhibiting substances for spiking of a surface water sample with a concentration of 10 ng/μL of each substance was prepared.

Chromatogram layer

HPTLC LiChrospher silica gel 60 F_254S plates (Merck) were immersed twice in 2-propanol for 20 min. After drying at 120 °C the plates were predeveloped to the top edge with acetonitrile. Finally, the plates were heated again to 120 °C for 20 min.

Sample application

Bandwise application with Automatic TLC Sampler (ATS 4), band length 6 mm, distance from the lower edge 8 mm, distance from left edge 20 mm, application volume for standard solutions and spiked surface water sample 10 μL (first development) and 200 μL of collected fractions (second development)

Chromatography

First development is done in the AMD 2 with gradient (16 steps) up to a total migration distance of 80 mm. Isocratic development in the 2nd dimension is performed in the Automatic Development Chamber (ADC 2) with chamber saturation (15 min). The plate is activated for 1 min with a molecular sieve, preconditioned for 30 s, developed up to 70 mm and then dried (20 min with cold air).

AChE inhibition assay

The pH was adjusted to 7.5 by placing the HPTLC-plate in a NH₃ vapor saturated twin through chamber, followed by drying with vacuum for 10 min. Then the plate was immersed with the Chromatogram Immersion Device in the AChE solution (0.05 M Tris-HCl buffer, pH 7.8, with 0.1 % bovine serum albumin), dipping speed 2 cm/s, dipping time 2 s. After subtraction of the abundant solution, incubation of the plate (5 min, 37 °C, > 90 % humidity) follows. After 6.5 min the substrate (0.5 g/L 3-indoxyl-3-acetate in aqueous solution with 4 % DMSO) was applied (amount of reagent 580 μL, distribution 0.03 μL/mm²) by spraying. Through active enzyme the substrate is cleaved to indoxyl, which reacts with oxygen to indigo white.

Documentation

With the TLC Visualizer 10 pictures (1 picture/min, exposure time 800 ms) are taken under UV 366 nm. For the evaluation of the effect, the picture after 2 min was used.

Elution from the plate

With the TLC-MS Interface the zone is eluted with methanol for 1 min with a flow rate of 0.2 mL/min

Results and discussion

The first separation was performed in the AMD system with a 16-step gradient consisting of methanol (0.05 % formic acid), dichloromethane and n-hexane – achieving a peak capacity of 30. With the development of a dynamic heart cut 2D separation approach the peak capacity was increased. Therefore, the first AMD chromatogram was parted in five equal hR_F-ranges. One effective substance was selected and the respective area investigated.

Based on 35 model substances the improvement of separation efficiency was shown. For example, it was possible to distribute seven substances, which occur in the first dimension in the hR_F-range of 80–100, through the second dimension to the hR_F-range of 7–91.

After method development the peak capacity of this dynamic approach was determined based on 125 substances. Therewith a peak capacity of 204 could be achieved, so that separation efficiency could be improved through the factor 7 in comparison to the 1D. The developed approach was applied to 50 AChE-inhibiting substances spiked with surface water sample. After AMD separation and the AChE 4inhibition assay in the 1D seven effective zones were selected. These were eluted and separated in the 2D with respect to the hR_F-range adapted mobile phase. With the AChE inhibition assay of the 2D, induced through increased peak capacity, obviously more effective zones could be detected compared to 1D (17 as opposed to 7). More- over it was possible to assign 8 of the effective chromatogram zones clearly to one substance, based on their hR_F-values. After 1D the assignment succeeded only for 2 zones.

Through the developed dynamic 2D approach, the peak capacity of EDA could be increased by the factor 7, which enhanced assignment of substances in the spiked surface water. The required time of 2D-EDA of the spiked surface water sample amounted to 8 h. To identify also unknown effective substances in samples, a coupling of this method to further analytical techniques, like MS is possible. Through identification of effective substances, their relevance can be easier evaluated, which is also useful for determination of limit values.

01

AMD gradient consisting of methanol – formic acid 100:0,05, dichloromethane and n-hexane [2]
02

Image of the dynamic heart cut 2D approach with elution of the chromatogram zone from the parallel partial HPTLC plate that was only developed (no AChE assay) [2]
03

Spreading of the hR_F-range in the 2D, depicted based on 35 model substances [2]
04

Application of the approach for EDA of a spiked surface water sample

Literature

[1] Weber, W. H., et al. CBS 94 (2005) 2
[2] Stütz, L., et al. Journal of Chromatography A 1524 (2017) 273

Further information is available from the authors on request.

Contact: Lena Stütz, Laboratory for Operation control and Research, Zweckverband Landeswasserversorgung, Am Spitzigen Berg 1, 89129 Langenau, Germany, Stuetz.L@lw-online.de

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Cleaning Validation in API Production Units

Cleaning validation at API production units

Written by Admin Camag on July 22, 2019. Posted in Case Studies.

HPTLC offers an inexpensive and time-saving way to control industrial cleaning. The following describes the procedure.

Specifications for cleaning validation:

1. Choice of cleaning solvent

The solubility of the manufactured products in different solvents is investigated. Solvents of low toxicity are preferred like acetone, ethanol, 2-propanol or acetic acid.

2. MACO calculation

The calculation takes into account the therapeutic daily dose (TDD) of active ingredients (AIs) as well as a factor of security depending on the route of administration of the product B.

MACO = TDD A x 10⁶ / Sf x TDD B

MACO in mg/kg (maximum allowable carryover)
TDD A in mg/kg (cleaned product)
TDD B in mg/kg (following product)
Safety factor (Sf) depending on the route of administration

Route of administration	Sf
Use externally	10
Oral product	100
Injectable product	1000

3. Calculation of specified concentration

Corresponds to the concentration of product tolerated in the last rinsing solvent. It is define on the basis of MACO, the batch size and the volume of cleaning solvent.

Specification = MACO x Sb / V

Specification in mg/L
MACO in mg/kg
Size of product lot (Sb, in kg) manufactured after cleaning (product B)
Volume (V, in L) of the last solvent rinsing to clean

Validation and control of industrial cleanings

1. Sample preparation

Industrial installations are cleaned by successive fillings with cleaning solvent. The cleaning is completed by a mechanical action. The sample (250 mL) is directly levied at the time of the emptying of the equipment and used for HPTLC without any sample preparation. The specification (maximum acceptable concentration) is calculated on the basis of the MACO and the volume of the equipment.

2. Sample application

In the ADC 2 with chamber saturation (usually for 3 min), migration distance 50 mm from lower plate edge. Prior to development, plates are conditioned at 33 % relative humidity for 5 min using a saturated solution of magnesium chloride. The mobile phase depends on the method selected due to the different polarities of the compounds.

3. Chromatography

Bandwise with Automatic TLC Sampler (ATS 4), 15 tracks, band length 8 mm, track distance 11.4 mm, distance from the side 20 mm, distance from lower edge 8 mm, usual application volumes between 0.5 and 2 µL for standard solutions and between 0.5 and 100 µL for samples solutions according to the respective specification.

4. Densitometry

TLC Scanner 4 with visionCATS/winCATS software, spectra recording from 200 to 700 nm for identification, quantification by absorption measurement at the specific wavelength for each target analyte, slit dimension 5.00 x 0.45 mm, scanning speed 20 mm/s, polynomial calibration by peak height.

5. Results

The conformity of a cleaning is assessed on two criteria: 1) The last 3 rinses are controlled to verify the effectiveness of the cleaning. This efficiency is assessed and evident by the gradual decrease of the residual product concentration in the different samples 2) In the last rinse, the residual product concentration in the sample has to be below the respective specification. Each sequence consists of three samples per cleaning, one sample of solvent of “white” rinse (the solvent blank used for the last rinsing) and standards. Three standard levels are used to facilitate the assessment, i.e. the identification of the target substances (the AIs) and evaluation of compliance: one standard level corresponding to: the limit of quantification (T3) the specification (T2) twice the specification (T1).

01

Example of a densitogram at 230 nm for cleaning validation
02

Analytical results recorded

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Adulteration of Ginkgo Supplements and Quality Issues

Adulteration and poor quality of Ginkgo biloba supplements

Written by Admin Camag on July 22, 2019. Posted in Scientific Publications.

Abstract

Adulteration of Ginkgo products sold as unregistered supplements within the very large market of Ginkgo products (reputedly £650 million annually) through the post-extraction addition of cheaper (e.g. buckwheat derived) rutin is suspected to allow sub-standard products to appear satisfactory to third parties, e.g. secondary buyers along the value chain or any regulatory authorities. This study was therefore carried out to identify products that did not conform to their label specification and may have been actively adulterated to enable access to the global markets.

500 MHz Bruker NMR spectroscopy instrumentation combined with Topspin version 3.2 and a CAMAG HPTLC system (HPTLC Association for the analysis of Ginkgo biloba leaf) were used to generate NMR spectra (focusing on the 6–8 ppm region for analysis) and chromatograms, respectively.

Out of the 35 samples of Ginkgo biloba analysed, 33 were found to contain elevated levels of rutin and/or quercetin, or low levels of Ginkgo metabolites when compared with the reference samples. Samples with disproportional levels of rutin or quercetin compared with other gingko metabolites are likely to be adulterated, either by accident or intentionally, and those samples with low or non-existent gingko metabolite content may have been produced using poor extraction techniques. Only two of the investigated samples were found to match with the High-Performance Thin-Layer Chromatography (HPTLC) fingerprint of the selected reference material. All others deviated significantly. One product contained a 5-hydroxytryptophan derivative, which is not a natural constituent of Ginkgo biloba.

Overall, these examples either suggest a poor extraction technique or deliberate adulteration along the value chain. Investigating the ratio of different flavonoids e.g. quercetin and kaempferol using NMR spectroscopy and HPTLC will provide further evidence as to the degree and kind of adulteration of Gingko supplements. From a consumer perspective the equivalence in identity and overall quality of the products needs to be guaranteed for supplements too and not only for products produced according to a quality standard or pharmacopoeial monograph.

https://www.sciencedirect.com/science/article/pii/S2210803316300239

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Comprehensive HPTLC Fingerprinting for Ginkgo Purity

Comprehensive HPTLC fingerprinting as a tool for a simplified analysis of purity of ginkgo products

Written by Admin Camag on July 19, 2019. Posted in Scientific Publications.

Abstract

Ethnopharmacological relevance

Herbal medicinal products based on ginkgo leaf refined dry extract (GBE) are an European development from the Eastern Asia traditionally used species Ginkgo biloba L. Nowadays, ginkgo products have increased the presence in the market, mainly as dietary supplements. Its adulteration with rutin and quercetin or herbal extracts rich in these compounds is a common practice. Tests featuring assays and detection of adulterants need to be performed on top of other existent methods (e.g. identification test). This may increase the costs of evaluating the quality of ginkgo products.

Aim of the study

To prove that comprehensive HPTLC fingerprinting can provide information beyond identification of ginkgo products, avoiding additional chromatographic tests for detection of adulterations.

Materials and methods

The information contained in the fingerprint obtained by HPTLC analysis of flavonoids was used for identification and for detection of adulterants, as well as to verify the limits of rutin and quercetin, which are normally determined by HPLC and used for detection of adulterants. For this purpose, peak profiles were generated from HPTLC chromatogram images. USP-HPLC methods were used for quantification of total flavonoids and testing the limits of rutin and quercetin. HPLC data were used to support the validity of the HPTLC method. An additional reversed phase HPTLC method was developed as a possible confirmatory method for the quercetin limit test.

Results

The proposed HPTLC method uses a particular sequence of detections, resulting in a number of images, which are later interpreted in a certain order. It is able to identify ginkgo products, to detect adulterants (rutin, quercetin, sophora fruit and flower bud, and buckwheat), and, using peak profiles generated from the chromatogram images prior to and after derivatisation, to evaluate the limits of rutin and quercetin. Forty-eight out of fifty-nine ginkgo dietary supplements analysed contained one or more adulterants. Furthermore, results of the HPTLC and HPLC limit tests for rutin and quercetin were in agreement in 98% of the cases. Finally, a decision tree showing the sequence of interpretation of the fingerprints obtained with the different detections after a single HPTLC analysis is included to help the analyst to evaluate whether samples have the correct identity and whether they contain or not adulterants.

Conclusion

A single HPTLC analysis is able to provide information on identity and purity of the products. This simplifies the analytical workflow and reduces the number of analyses prescribed in the USP powdered ginkgo extract monograph.

https://www.sciencedirect.com/science/article/pii/S0378874118345410

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HPTLC Screening for Undeclared Drugs in Weight Loss Products

HPTLC Screening of Weight Loss Products for Undeclared Synthetic Drugs

Written by Admin Camag on July 19, 2019. Posted in Scientific Publications.

Abstract

Background

The proposed HPTLC method combines features of the existing methods for (1) the detection of sibutramine and (2) for the detection of phosphodiesterase type 5 inhibitors and analogs.

Objective

The method permits effective screening for the presence of nine adulterants in finished products, including tablets, capsules, and “instant coffee” powders.

Methods

All products were prepared for analysis using the same simple procedure: ultrasound-assisted extraction in methanol for 30 min followed by centrifugation or filtration.

Results

The retardation factor (RF) values of individual zones afford preliminary identification of potential adulterants. Scanning densitometry enables comparison of recorded UV spectra with those of known standard compounds and provides further structural information.

Conclusions

The method was successfully applied to 12 commercial products. Of those, nine products tested positive for at least one undeclared component.

https://www.ncbi.nlm.nih.gov/pubmed/30454078

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HPTLC Method for Caffeine Identification in Herbal Drugs

A harmonized HPTLC method for identification of various caffeine containing herbal drugs

Written by Admin Camag on July 19, 2019. Posted in Scientific Publications.

Abstract

Caffeine is an alkaloid used for long times in food, beverages, medicines, dietary supplements, and in botanicals such as coffee, cacao, cola, guarana, or tea. Today, more and more products are made with caffeine rich extracts from natural sources, or by adding caffeine. In order to prevent confusion about the origin of a caffeine-containing product, a specific method is needed for the determination of the botanical or chemical origin of the caffeine. As caffeine is known to be a central nervous system stimulant that can affect blood pressure and heart rate, all countries in the European Union require drinks (except tea and coffee) with more than 150 mg/L caffeine content to be labeled “high caffeine content”, followed by their caffeine content expressed in mg/100 mL. The United States Department of Agriculture publishes food composition data including levels of caffeine in foods. Until now, there is no regulation for labeling the caffeine content of dietary supplements. This paper describes the development of a harmonized HPTLC method for the determination of the botanical origin of caffeine containing extract, and the detection of added caffeine in cases of adulteration. After identification, the verification of the caffeine content is necessary. In the absence of specific label information, the proposed method can be used to estimate the caffeine content of a product and where specific label information is present, it provides a limit test.

https://www.tandfonline.com/doi/abs/10.1080/10826076.2019.1585612?journalCode=ljlc20

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Quantification of Bitter Acids in Hops

Written by Admin Camag on July 19, 2019. Posted in CBS Articles.

TLC has a wide range of qualitative and quantitative application fields. The Instrumental Analytics group of Michael Schulz at Merck in Darmstadt, Germany, develops new chromatographic TLC and HPTLC layers. In this context the group works on new application fields for the use of these techniques.

Introduction

The amount of bitter acids in hops is a very important parameter for beer production. Different types of hops contain different quantities of α- and ß-acids responsible for the bitterness of the beer. In general hops are divided into aromatic hops (<10% α-acids) and bitter hops (>10% α-acids).

In this study, a total of 12 samples were analyzed, i.e. four aromatic hops, four bitter hops and four hops of the same variety, but from different regions. The sample extracts were applied on HPTLC plates silica gel 60 F254 MS-grade, developed and separated with a gradient using the Automated Multiple Development (AMD 2) System and their fluorescence was measured at 360/>400 nm.

Sample preparation

Crumbled hop pellets (5 g) suspended in 10 mL methanol, 50 mL diethyl ether and 20 mL 0.1 M hydrochloric acid solution were stirred for 40 min. 2 mL of the upper ether phase were transferred to a 20-mL volumetric flask and filled up to the mark with methanol (10 mg/mL). An aliquot of this extract was filtered via a 0.45-μm membrane filter into a sampler vial.

Standard solution

The International Calibration Extract (ICE) 3 contained 44.6% α-acids, 24.3% ß-acids and 31.1% other ingredients dissolved in methanol (1 mg/mL).

Chromatogram layer

HPTLC plate silica gel 60 F₂₅₄ MS-grade, 20 x 10 cm (Merck)

Note: HPTLC plates for mass spectrometry are characterized by a higher purity of the layer and a thinner layer thickness (100 μm). This allows a more sensitive measurement compared to the standard plate. The suitability for MS is also shown in the certificates of analysis.

Sample application

With Automatic TLC Sampler (ATS 4) as 6 mm bands, application volumes were 0.5–1.6 μL/band for standard and 0.3–1.0 μL/band for sample solutions.

Chromatography

Automated multiple development using a 9-step AMD 2 gradient based on ethyl acetate – methanol – n-heptane followed by drying for 2 min after each step

Densitometry and documentation

Results and discussion

Using an AMD 2 gradient separation on HPTLC plates MS-grade, it was possible to separate the α-acids (hR_F 36) and ß-acids (hR_F 65) from the hop matrix, showing the difference in the bitter acid content in the various hops. Although hops contain a high amount of matrix, both types of bitter acids were well separated and quantified by fluorescence measurement at 360/>400 nm. With this study the differences in the bitter acid content of both regional and varietal hops was determined. In most cases, the bitter hops contained considerably more bitter acids than aromatic hops. The content of bitter acids differed for different regional origins of the same varietal hop.

01

9-step AMD 2 gradient based on ethyl acetate
02

AMD 2 chromatogram at UV 366 nm of ICE 3 (three different volumes) and the 4 aromatic hops (no. 1–4, threefold applied n = 3)
03

Fluorescence measurement at 360/>400 nm

Determined content of bitter acids in 12 hops

Literature

[1] Adrian Forster et al. (2012). Hopfen. Vom Anbau bis zum Bier, Carl, Nürnberg
[2] B. Engelhard, A. Lutz, E. Seigner (2011). Hopfen für alle Biere der Welt, www.lfl.bayern.de

Further information on request from the authors.

Contact: Michael Schulz, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany, michael.schulz@merckgroup.com

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Identification of fixed oils by HPTLC

Written by Admin Camag on March 19, 2019. Posted in Case Studies.

This case study demonstrates a rapid identification of fixed oils after derivatization with phosphomolybdic acid.

Introduction

HPTLC is a powerful technique with regard to a high sample throughput capacity, short analysis time, and low running costs. Our case study demonstrates a rapid identification of fixed oils after derivatization with phosphomolybdic acid.

Challenge

Often chromatographic techniques require a time- and cost-intensive sample preparation step. In many cases HPTLC allows to analyze several samples side by side after only a minimal sample preparation (e.g. dissolving). The identity of samples with unknown composition can be elucidated by comparing the pattern of the sample with that of reference materials. A System Suitability Test (SST) can be applied to check the reproducibility of the chromatographic system during method transfer validation and to qualify the data on each plate.

Description of the procedure

The method is downloaded from the visionCATS Method Library. The Word document (SOP) is opened for reference. In preparation for the analysis a RP-18 HPTLC plate is pre-washed with dichloromethane and then dried in the oven. All samples and references are prepared according to the SOP (in compliance with the USP method <203>, Application note F-39). The visionCATS method file is opened and executed, creating a new analysis file. In this visionCATS analysis file the sequence table is completed (Vial ID, description of samples and references, and selection of the rack position of each vial). An image of the pre-washed clean plate under white light is captured with the TLC Visualizer. The samples and standards are applied band-wise onto the HPTLC plate using the ATS 4, which guarantees precise and reliable application. Then the HPTLC plate is developed using the ADC 2. For visualization of fingerprints of the different fixed oils the developed plate is dipped into phosphomolybdic acid reagent, heated at 120°C for 3 minutes, and documented with the TLC Visualizer under white light. For a successful method transfer validation the SST needs to pass. If the RF values are in the specified range the patterns of the unknown samples and those of the references can be easily compared with the visionCATS Image Comparison.

Image Comparison of fixed oils and references (USP) after derivatization

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.
HPTLC is also used for determination of purity (adulteration/fraud) and stability studies (shelf life).
HPTLC can be used as high throughput and low-cost technique for the analysis of food samples.

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