Review on Hyphenation in HPTLC-MS

 

R. P. Bhole*, S. R. Jagtap, K. B. Chadar, Y. B. Zambare

Department of Pharmaceutical Quality Assurance, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune- 411018 Maharashtra, India

 

ABSTRACT:

TLC-MS is a versatile technique for separation as well as identification of Pharmaceuticals and Phytopharmaceuticals. Traditionally the separation was carried out by TLC/HPTLC then the separated materials was removed and then identified by Mass spectrometry. Many hyphenated techniques like HPTLC-MS, GC-MS, HPLC-MS, MS-MS Etc. are available. HPLC-MS is more preferred method for separation and identification of compounds. But disadvantage of HPLC is that it requires more solvent as compared to HPTLC. This technique provides efficient, quick and simple method for identification and separation of Narcotic drugs and psychotropic substances. Therefore taking advantage of less solvent requirement in HPTLC and also to enhance working hyphenation of both HPTLC and MS is done so as to provide wide scope for separation as well as identification of product within short period of time. The great advantage of the instrument is that exclusively questioned zones are transferred into the MS for identification and that within less than one-minute sensitive mass spectrometric information is available. This review includes Introduction of method, information of HPTLC and ms, interface for HPTLC-MS, Advantage with example and Applications of method.

 

 

INTRODUCTION:

Introduction high performance thin layer chromatography (HPTLC)^(1-7):

Thin layer chromatography is the basic planar chromatography method is used for the separate the volatile and non volatile substances. High performance thin layer chromatographic method was developed for estimation of crude drug, synthetic, and semi synthetic drug. Chromatographic technique is used to separate volatile as well as non-volatile compound (mixtures). This chromatography performed on the sheet of aluminum foil, glass or plastic which is coated with a thin layer of adsorbent material usually silica gel, cellulose and aluminum oxide. Adsorbent layer is called as a stationary phase (S.P.).

 

Applied sample on the plate by injection, a solvent or solvent mixture (mobile phase) is drawn up the plate via capillary action. Because different compounds having different separation time. Also requirement of different mobile phase for different mixture or drug. Different rates of separation are archived by TLC technique.

 

Chromatographic technique can be used to monitor reaction, identify compounds present in a given mixture and determine the purity of a compound. Specific examples of these applications include analyzing crude drug, detection of impurities in compound, pesticides or insecticides from food, radiochemical purity from radiopharmaceuticals, medicinal constituents in the plants (herbal) and water, detection of the dye composition from fibers in forensics.

 

Separation of compounds depends on the competition of the compound (solute), mobile phase and stationary phase. silica gel is used in normal phase chromatography as a stationary phase it is polar in nature. Two or more than two elute present in the mixture which different polarity. Those more polar elute last and less polar elute first. The more polar compound has a stronger interaction with the silica and is therefore more capable to disappear the mobile phase from the binding places. Less polar solvent or mobile phase gives higher R_f value. If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of disappearing solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate and R_f value not more than one. It is commonly said that "strong" solvents (eluents) push the analyzed compounds up the plate, while "weak" eluents only move them.

 

Basic principle of HPTLC^(6,11-13):

Similar to other chromatographic methods, thin layer chromatography is also based on the principle of separation.

i.      The separation depends on the relative affinity of compounds towards stationary and the mobile phase.

ii.    The compounds under the influence of the mobile phase (driven by capillary action) travel over the surface of the stationary phase. During this movement, the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus, separation of components in the mixture is achieved.

iii.  Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character are identified by means of suitable detection techniques.

 

Table 1:- Trouble Shooting In HPTLC

Sr. No.	Cause	Remedy
1.	Poor band quality (Linomat applicator) Gas flow not optimal. Wrong distance between needle tip/ TLC layer Damaged or clogged needle.	a)  Check gas pressure and adjust to 2-3 bars. b)   Check distance and adjust to 1 mm. c)  Remove syringe and fill it with solvent. Force out by hand.
2.	Bad reproducibility	Limit switch for sample dosage syringe misadjusted.
3.	Poor accuracy (linomat applicator). a)  Damaged spray head due to wrong sample dosage syringe type. b)  Leaky syringe because of destroyed glass barrel.	a)  Replace the spray head. b)  It happen because of high pressure, replace the sample dosage syringe.
4.	Poor band quality a)  Check gas supply for spraying. b)  Check nozzle for clogs. c)  Check distance capillary/nozzle and capillary/object. d)  Application position is overloaded with sample.	a)  Check the regulating gas flow. b)  Check cleaning the spray nozzle. c)  Check adjusting the capillary d)   Dissolve sample in more suitable solvent.
5.	Leakage a)  If air bubbles are formed, when the syringe piston has a leak. b)  Another reason for leaks could be caused by the O-ring gasket in the connection between syringe and capillary.	Check and replacing the o-ring (gasket) as well as syringe piston.

 

Mass spectroscopy^{(6, 8, 9, 10)}:

Mass spectrometry (MS) is a powerful method for identifying complex mixtures. Mass spectrometric methods have always put strong controls on the geometry and the preparation of samples. This method is used to find the composition of a physical sample by generation a mass spectrum representing the masses of samples. Spectra are used to determine the isotopic or elemental signature of a sample, the masses of particles, molecules, and to elucidate the chemical structures of compounds, such as peptides and other chemical compounds. Mass spectrometry (MS) is an analytical technique that produces spectra of the masses of the atoms or drug molecules comprising a sample of material. Mass spectrometry works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratio.

 

General procedure of mass spectrometry, a sample, which may be solid, liquid, or gas is ionized (compounds or molecules get vaporized for example by bombarding electrons on sample). This may cause some of the sample's molecules to break/formation into charged fragments (ions). These ions are then separated according to their mass-to-charge ratio by using electric or magnetic field or typically by accelerating them. Fragments of the Ions same mass-to-charge ratio will undergo the same amount of deflection. The Ions are detected by a mechanism capable of detecting charged particles, by electron multiplier. Results spectra of the relative abundance detected ions as a function of the mass-to-charge ratio. The molecules or atoms in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.

 

MS include molecular weight characterization, non covalent interactions, protein and peptide sequencing, DNA sequencing, protein folding, in vitro drug analysis, and drug discovery.

Introduction of MS^(6,18-22):

The instrument consists of three major components.

a)     Ion Source:- For producing gaseous ions from the substance being studied.

b)    Analyzer:- For resolving the ions into their characteristics mass components according to their mass-to-charge ratio.

c)     Detector System:- detecting the ions and recording the relative abundance of each of the resolved ionic species.

 

A sample introduction system is necessary to introduce the samples to be studied to the ion source while maintaining the high vacuum requirements (10-6 to 10-8 mm of mercury). Computer and A techniques are required to control the instrument, acquire and manipulate data and compare spectra to reference.

 

Mass spectrometry used ^{(23-29, 6)}:

Sample is introduced into the mass spectrometer and the molecules are ionized and accelerated. The Ions are separated by mass and charge by the mass analyzer via electromagnetic deflection and the ions then detected and amplified. The entire system is under intense vacuum during the entire procedure. After signal amplification, the ion hits are analyzed, and data is generated that yields the relative abundance of each ion based on the mass-to-charge (m/z) ratio. Although sector instruments have decreased in use in recent years due to improvements in mass analyzers (e.g. quadrupole, ion trap, TOF) the simplified diagram conveys a key principle of mass spectrometry. The ability to select and analyze specific ions in a complex sample.

·       To identify unknown compounds

·       To quantify known materials

·       To elucidate the structural and chemical properties of molecules

·       Detection of compounds at 10-12g, 10-15 moles for a compound of mass 100 Dalton.

 

Purpose:

·       Biotechnology:- analysis of proteins, peptides, oligo nucleotides.

·       Pharmaceutical Analysis:- drugs discovery, combinatorial chemistry, pharmokietics, drug metabolism.

·       Clinical Examination:-neonatal screening, haemoglobin analysis, drug testing.

·       Environmental Analysis:- water, food, air quality (PCBs etc).

·       Geological Analysis :- oil composition.

 

Interfaces^(30-33):

High performance thin layer chromatography (Camag, Muttanz, Switzerland) consisted of an auto sample applicator Linomat V connected to a nitrogen cylinder, TLC scanner attached to a PC running win-CATS software (version 1.4.4), TLC Visualizer, and Camag twin-trough chambers were used in analysis. HPTLC-MS detailed examination was carried out by TLC-MS interface using acetonitrile as eluting agent at a flow rate of 1 ml/min. It remove/take out circular zones in the form of bands from the developed HPTLC plate. The eluted material was transferred automatically to single-quadrupole mass spectrometer, and mass spectra was recorded. Surveys have shown that not all samples may be processed by HPTLC-MS or HPTLC-DAD or HPTLC-MALDI or low delectability of the compounds or impurities in the UV range, a heavy matrix load or a deficiency of MS compatible solvents. On the other hand HPTLC is another very fast and suitable method to separate samples. In the past unknown substances were scraped off from the TLC/HPTLC plate, eluted into a tube with solvent and transferred into the MS. Now a very suitable and universal TLC-MS Interface is available which can semi-automatically extract zones of interest and direct them online into any brand of HPLC-MS system. The interface is fast and simply connected (by two fittings) to any LC-coupled mass spectrometer without adjustments or mass spectrometer modifications. Questioned materials are directly extracted from a TLC/HPTLC plate and sensitive mass spectrometric signals are obtained within a minute per substance zone. The interface extracts the complete material zone with its depth profile and thus allows detections comparable to HPLC down to the pg/zone range. The interface has been demonstrate to be one of the most reliable and versatile interfaces for TLC/HPTLC-MS coupling.

 

Principle of HPTLC-MS^(34-38):

The versatile instrument is used to isolate unknown compounds from a HPTLC/TLC plate and transfer them into a mass spectrometer for identification or structure elucidation. TLC/MS Interface can be bring together to any brand of LC-coupled mass spectrometer. Plugand play installation by two HPLC fittings at a given HPLC-MS system Semi-automatic instrument involving automatic piston movement for pressure seal the HPTLC/TLC zone on both glass plates and aluminum foils take out directly from the plate using a suitable solvent delivered by the HPLC pump Online transfer into the mass spectrometer. Automatic cleaning of the piston between the extractions.

 

Key features^(22-28):

·       Rapid and contamination-free elution of selected zones.

·       Online transfer into the mass spectrometer.

·       Compatible with all conventional HPLC-MS systems.

·       Identification of unknown substances at a limit of detection as known from HPLC-MS.

 

Low solvent consumption:

 

Information of HPTLC-MS^(12-18):

Materials:

Plates or aluminum foils up to 20 x 20 cm can be positioned precisely and analyzed zone by zone.

Automation:

Semi-automatic instrument including automatic piston movement, automatic cleaning of the piston, manual positioning and switching.

Weight:

around 11 kg

Size:

around 23 x 50 x 25 cm with optional size

large table is 40 x 50 x 25 cm (w x d x h)

Requirements:

5 bar compressed air or N2, HPLC pump or HPLC-MS system.

 

Structure identification and elucidation^(39-43):

Following proper isolation using proper chromatographic technique, the next step is the characterization and elucidation of the unknown isolated bioactive materials. Typically, hyphenation is selected to provide the most relevant information as mandatory. In this case spectra can be recorded directly from zones of interest on an HPTLC plate through the flexible TLC-MS interface. Nuclear Magnetic Resonance (NMR) has been connected with several bio autography. Even on analytical range, it has been applied for structure confirmation. Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy is also a well-known method for characterization. It has discovered some application in natural product research and has been attached with chromatography. Surface Enhanced Raman Spectroscopy (SERS) has also been connected with chromatography and could find some applications in natural product research. UV/VIS/FLD spectrometry connected with chromatography is also a useful method in bioactive materials identification. It is usually coupled with planar chromatography from which images can be grabs directly on the plate. Most of these experiments can be calculated directly from analytical plates. Important samples are introduced directed to mass spectrometer (MS) or high-resolution mass spectrometer (HRMS) for structure confirmation.

 

CONCLUSION:

1.     This above short report a simple hptlc-ms method for the the routine analysis of pharmaceutical product/formulation.

2.     Its play important role in the study of the drug metabolism, discovery of new drug candidate and the analysis, identification and characterization of impurities and degradent in drug substants.

3.     With regard to the outcome of this study and recommendations for plate handling, special plate forms are rational.

4.     The hptlc-ms interface is versatile building block of multidimensional liquid chromatographic system.

5.     Lc-ms have proved to be an extremely sensitive and specific technique for the analysis of pharmaceutical product.

 

Application:

1. The liquid chromatography–mass spectrometry study of degraded products gave the idea about the fragmentation patterns of degraded products. The developed method can be used for routine analysis of terizidone and its formulations

 

 

Table 2: Results of forced degradation study

 

 

2. Degradation product of Empagliflozin in alkaline condition was carried out and its degradation product is successfully separated and isolated by HPTLC method. degradation product was identified by using MS-MS technique.

 

Table 3 :- Results of forced degradation studies

Stress condition	Assay of active substance	Rf values of degraded products
Acid (0.1 M HCl)	82.74	0.16, 0.32, 0.66
Base (0.1 M NaOH)	80.04	0.13, 0.25, 0.53, 0.67
Oxide (3 % H2O2)	82.92	0.24, 0.31, 0.65
Neutral (Distilled water)	77.89	0.27, 0.65
Thermal	73.80	0.27, 0.47, 0.67
UV degradation	83.23	0.32, 0.55, 0.65

 

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Accepted on 31.07.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):1028-1034.