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Comprehensive HPTLC fingerprinting for the quality control of Angelica gigas root

HPTLC Fingerprinting for Angelica Gigas
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 F254s (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


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

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

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)

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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)

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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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Comprehensive HPTLC fingerprinting for the quality control of Angelica gigas root

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