Automated Post-Column Sample Manipulation Prior to Detection in Liquid Chromatography: A Review of Pharmaceutical and Bioanalytical Applications

Author(s): Natalia Manousi, Constantinos K. Zacharis*.

Journal Name: Current Analytical Chemistry

Volume 15 , Issue 7 , 2019

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Graphical Abstract:


Automated post-column sample manipulation is undoubtedly one of the most challenging approaches in liquid chromatography for the improvement of method selectivity and sensitivity. With the post-column analyte derivatization being the most-abundant approach approach of this category, other strategies typically comprise post-column infusion of internal standard or other reagents prior to mass spectrometric detection to enhance the ionization efficiency of the analyte or to compensate the ion suppression/enhancement.

In this review, on-line post column methodologies focused on the quality control of pharmaceuticals and biomedical applications will be presented and discussed. Emphasis will be given on the automation capabilities of such systems.

Keywords: Automation, bioanalysis, derivatization, infusion, internal standard, liquid chromatography, pharmaceuticals, postcolumn.

Kazakevich, Y.; LoBrutto, R. HPLC for Pharmaceutical Scientists, 2006.
Guillarme, D.; Veuthey, J-L. UHPLC in Life Sciences; Guillarme, D., Veuthey, J.-L. , Eds.; RSC Chromatography Monographs; Royal Society of Chemistry: Cambridge; , 2012.
[ 9781849735490]
Gosetti, F.; Mazzucco, E.; Zampieri, D.; Gennaro, M.C. Signal suppression/enhancement in high-performance liquid chroma-tography tandem mass spectrometry. J. Chromatogr. A, 2010, 1217(25), 3929-3937.
Van Eeckhaut, A.; Lanckmans, K.; Sarre, S.; Smolders, I.; Michotte, Y. Validation of bioanalytical LC-MS/MS assays: Evaluation of matrix effects. J. Chromatogr. B , 2009, 877(23), 2198-2207.
Chan, C-C.; Bolgar, M.S.; Dalpathado, D.; Lloyd, D.K. Mitiga-tion of signal suppression caused by the use of trifluoroacetic acid in liquid chromatography mobile phases during liquid chromatography/mass spectrometry analysis via post-column addition of ammonium hydroxide. Rapid Commun. Mass Spectrom., 2012, 26(12), 1507-1514.
Zheng, S.; Ma, Z.; Han, H.; Ye, J.; Wang, R.; Cai, S.; Zhou, H.; Yu, L.; Zeng, S.; Jiang, H. Post-Column mobile phase adjustment: a strategy to eliminate the contradiction between liquid chromatography and mass spectrometry in the determination of flavonoids in rat plasma. J. Pharm. Biomed. Anal., 2014, 95, 176-183.
Ouyang, L-F.; Wang, Z-L.; Dai, J-G.; Chen, L.; Zhao, Y-N. Determination of total ginsenosides in ginseng extracts using charged aerosol detection with post-column compensation of the gradient. Chin. J. Nat. Med., 2014, 12(11), 857-868.
Vancea, S.; Imre, S.; Donáth-Nagy, G.; Béla, T.; Nyulas, M.; Muntean, T.; Borka-Balás, R. Determination of free captopril in human plasma by liquid chromatography with mass spectro-metry detection. Talanta, 2009, 79(2), 436-441.
Rigas, P.G. Review: Liquid chromatography—post-column derivatization for amino acid analysis: Strategies, instrumentation, and applications. Instrum. Sci. Technol., 2012, 40(2-3), 161-193.
Poole, C.F. Derivatization in Liquid Chromatography. In: Liquid Chromatography; Elsevier Science B. V: Amsterdam, 2017, pp. 39-68.
Zacharis, C.K.; Tzanavaras, P.D. Liquid chromatography coupled to on-line post column derivatization for the determination of organic compounds: a review on instrumentation and chemistries. Anal. Chim. Acta, 2013, 798, 1-24.
Jones, A.; Pravadali-Cekic, S.; Dennis, G.R.; Shalliker, R.A. Post column derivatisation analyses review. is post-column deriva-tisation incompatible with modern HPLC columns? Anal. Chim. Acta, 2015, 889, 58-70.
Adegoke, O.A. An overview of applications of pre-column derivatization reactions for the liquid chromatographic analysis of pharmaceuticals and other compounds. Afr. J. Pure Appl. Chem., 2012, 6(14), 129-140.
Lawrence, J.F.; Frei, R.W. Chemical Derivatization in Liquid Chromatography. In: Journal of Chromatography Library. , Vol. 7; Elsevier: Amsterdam, 1985.
Chamkasem, N.; Cobb, W.Y.; Latimer, G.W.; Salinas, C.; Clement, B.A. Liquid chromatographic determination of Aflatoxins, Ochratoxin A, and Zearalenone in grains, oilseeds, and animal feeds by Post-Column derivatization and On-Line sample cleanup. J. Assoc. Off. Anal. Chem., 1989, 72(2), 336-341.
AOAC Official method 991.08: Glyphosate and Aminomethylphosphonic Acid (AMPA) in Environmental Water by Liquid Chromatographic Method.,
USEPA method 531.1: Measurement of N-Methylcarbamoyloximes and N- Methylcarbamates in Water by Direct Aqueous Injection HPLC with Post Column Derivatization.,
Application Note for Post-column Liquid Chromatography No. 102, Analysis of Biogenic Amines, Pickering Laboratories
Matsushita, K. Application note - automatic precolumn derivatization of amino acids and analysis by fast lc using the agilent 1290 infinity lc system, agilent technologies http:// Library/applications/5990-5599EN.pdf.
Spackman, D.H.; Stein, W.H.; Moore, S. Automatic recording apparatus for use in chromatography of amino acids. Anal. Chem., 1958, 30(7), 1190-1206.
Özyürek, M.; Baki, S.; Güngör, N.; Çelik, S.E.; Güçlü, K.; Apak, R. Determination of biothiols by a novel on-line HPLC-DTNB assay with post-column detection. Anal. Chim. Acta, 2012, 750, 173-181.
Miralles, P.; Chisvert, A.; Alonso, M.J.; Hernandorena, S.; Salvador, A. Determination of free formaldehyde in cosmetics containing formaldehyde-releasing preservatives by reversed-phase dispersive liquid-liquid microextraction and liquid chromatography with post-column derivatization. J. Chromatogr. A, 2018, 1543, 34-39.
Tzanavaras, P.D. A Green HPLC method for the determination of N-Acetylcysteine using post-column derivatization with methyl-propiolate. Instrum. Sci. Technol., 2012, 40(2-3), 150-160.
Zacharis, C.K.; Tzanavaras, P.D.; Themelis, D.G. Ethyl-Propiolate as a novel and promising analytical reagent for the derivatization of thiols: Study of the reaction under flow conditions. J. Pharm. Biomed. Anal., 2009, 50(3), 384-391.
Thirumangalathu, R.; Wong, K.N.; Coultas, J.; Hair, A.; Piedmonte, D.M. Implementation of a high-throughput ion chromatographic assay to assess glass degradation in drug product formulations. PDA J. Pharm. Sci. Technol., 2015, 69(1), 96-107.
Zuman, P. Reactions of orthophthalaldehyde with nucleophiles. Chem. Rev., 2004, 104(7), 3217-3238.
Douša, M.; Gibala, P.; Lemr, K. Liquid chromatographic separation of pregabalin and its possible impurities with fluorescence detection after postcolumn derivatization with O-Phtaldialdehyde. J. Pharm. Biomed. Anal., 2010, 53(3), 717-722.
Douša, M.; Břicháč, J.; Gibala, P.; Lehnert, P. Rapid hydrophilic interaction chromatography determination of lysine in pharmaceutical preparations with fluorescence detection after postcolumn derivatization with O-Phtaldialdehyde. J. Pharm. Biomed. Anal., 2011, 54(5), 972-978.
Douša, M.; Břicháč, J.; Svoboda, J.; Klvaňa, R. Rapid HILIC method with fluorescence detection using derivatization reaction utilizing O-Phthaldialdehyde for determination of degradation product of aliskiren. J. Pharm. Biomed. Anal., 2012, 66, 359-364.
Douša, M.; Pivoňková, V.; Sýkora, D. Optimization of o -Phtaldialdehyde/2-Mercaptoethanol postcolumn reaction for the hydrophilic interaction liquid chromatography determination of memantine utilizing a silica hydride stationary phase. J. Sep. Sci., 2016, 39(16), 3145-3155.
Douša, M. Quantification of 2-Aminoisobutyric acid impurity in enzalutamide bulk drug substance using hydrophilic interaction chromatography with fluorescence detection. J. Pharm. Biomed. Anal., 2019, 164, 296-301.
Zacharis, C.K.; Tzanavaras, P.D.; Vlessidis, A.G. Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on-line post-column derivatization. J. Sep. Sci., 2013, 36(11), 1720-1725.
Douša, M.; Stach, J.; Gibala, P.; Lemr, K. Quantification of structurally related aliphatic amino alcohols in l-Valinol by hydrophilic interaction liquid chromatography separation combined with postcolumn derivatization and fluo-rescence detection. J. Sep. Sci., 2016, 39(5), 851-856.
Douša, M.; Doubský, J. Separation of structurally related primary aliphatic amines using hydrophilic interaction chromatography withfluorescence detection after postcolumn derivatization with o - phthaldialdehyde/mercaptoethanol. J. Sep. Sci., 2017, 40(24), 4689-4699.
Yoshitake, M.; Nohta, H.; Ogata, S.; Todoroki, K.; Yoshida, H.; Yoshitake, T.; Yamaguchi, M. Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection. J. Chromatogr. B , 2007, 858(1-2), 307-312.
Dobashi, Y.; Santa, T.; Nakagomi, K.; Imai, K. An Automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction. Analyst , 2002, 127(1), 54-59.
Honda, S.; Matsuda, Y.; Takahashi, M.; Kakehi, K.; Ganno, S. Fluorimetric determination of reducing carbohydrates with 2-cyanoacetamide and application to automated analysis of carbohydrates as borate complexes. Anal. Chem., 1980, 52(7), 1079-1082.
Nohara, Y.; Suzuki, J.; Yamazaki, Y.; Kubo, H. Determination of idebenone in plasma by HPLC with post-column fluorescence derivatization using 2-cyanoacetamide. Chem. Pharm. Bull. , 2012, 60(5), 598-602.
Rybak, M.E.; Pfeiffer, C.M. Clinical analysis of vitamin B6: Determination of pyridoxal 5′-Phosphate and 4-Pyridoxic acid in human serum by reversed-phase high-performance liquid chromatography with chlorite postcolumn derivatization. Anal. Biochem., 2004, 333(2), 336-344.
Douša, M.; Břicháč, J.; Tkadlecová, M.; Man, S.; Zezula, J.; Hájíček, J.; Pekárek, T. A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent. J. Pharm. Biomed. Anal., 2016, 128, 391-397.
Sun, S.; Zhao, X.; Li, G.; Yu, G.; Xing, X.; Zeng, Y.; Wu, J.; Wang, J. Microanalysis of oligosaccharide HS203 in beagle dog plasma by postcolumn fluorescence derivatization method. Carbohydr. Polym., 2012, 89(2), 661-666.
Hirano, Y.; Tsunoda, M.; Funatsu, T.; Imai, K. Rapid assay for Catechol-O-Methyltransferase activity by high-performance liquid chromatography-fluorescence detection. J. Chromatogr. B , 2005, 819(1), 41-46.
Nakao, R.; Furutsuka, K.; Yamaguchi, M.; Suzuki, K. Sensitive determination of specific radioactivity of positron emission tomography radiopharmaceuticals by radio high-performance liquid chromatography with fluorescence detection. Nucl. Med. Biol., 2008, 35(7), 733-740.
Pérez-Ruiz, T.; Martínez-Lozano, C.; García-Martínez, M.D. A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates. J. Chromatogr. A, 2009, 1216(9), 1312-1318.
Lores, M.; Cabaleiro, O.; Cela, R. Post-Column photochemical derivatization in high-performance liquid chromatography. TrAC. Trends Analyt. Chem., 1999, 18(6), 392-400.
Cañada-Cañada, F.; Espinosa-Mansilla, A.; Muñoz de la Peña, A.; Mancha de Llanos, A. Determination of marker pteridins and biopterin reduced forms, tetrahydrobiopterin and dihydro-biopterin, in human urine, using a post-column photoinduced fluorescence liquid chromatographic derivatization method. Anal. Chim. Acta, 2009, 648(1), 113-122.
Woo, J.S.; Ryu, J.K. Quantitative determination of voglibose in pharmaceutical tablets using high-performance liquid chromatography-fluorescence detection with post-column derivatization and mass spectrometric detection. J. Pharm. Biomed. Anal., 2006, 42(3), 328-333.
Castillo-García, M.L.; Aguilar-Caballos, M.P.; Gómez-Hens, A. Determination of veterinary penicillin antibiotics by fast high-resolution liquid chromatography and luminescence detection. Talanta, 2017, 170, 343-349.
Taylor, P.J. Matrix effects: The achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry. Clin. Biochem., 2005, 38(4), 328-334.
Liang, Z. Perspectives on addressing ionization matrix effects in LC-MS bioanalysis. Bioanalysis, 2012, 4(10), 1227-1234.
Annesley, T.M. Ion suppression in mass spectrometry. Clin. Chem., 2003, 49(7), 1041-1044.
Matuszewski, B.K.; Constanzer, M.L.; Chavez-Eng, C.M. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC−MS/MS. Anal. Chem., 2003, 75(13), 3019-3030.
Choi, B.K.; Gusev, A.I.; Hercules, D.M. Postcolumn introduction of an internal standard for quantitative LC−MS analysis. Anal. Chem., 1999, 71(18), 4107-4110.
Stahnke, H.; Reemtsma, T.; Alder, L. Compensation of Matrix effects by postcolumn infusion of a monitor substance in multiresidue analysis with LC−MS/MS. Anal. Chem., 2009, 81(6), 2185-2192.
Loos, G.; Van Schepdael, A.; Cabooter, D. Quantitative mass spectrometry methods for pharmaceutical analysis. Phil. Trans. Math. Phys. Eng. Sci., 2016, 374(2079)20150366
Hall, T.G.; Smukste, I.; Bresciano, K.R.; Wang, Y.; McKearn, D.; Savage, R.E. Identifying and Overcoming Matrix Effects in Drug Discovery and Development. In: Tandem Mass Spectrometry - Applications and Principles; InTech, 2012.
King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. Mechanistic investigation of ionization suppression in electrospray ionization. J. Am. Soc. Mass Spectrom., 2000, 11(11), 942-950.
Petritis, K.; Valleix, A.; Elfakir, C.; Dreux, M. Simultaneous analysis of underivatized chiral amino acids by liquid chromatography-ionspray tandem mass spectrometry using a teicoplanin chiral stationary phase. J. Chromatogr. A, 2001, 913(1-2), 331-340.
Apffel, A.; Fischer, S.; Goldberg, G.; Goodley, P.C.; Kuhlmann, F.E. Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases. J. Chromatogr. A, 1995, 712(1), 177-190.
Silva, D.B.; Rodrigues, E.D.; da Silva, G.V.J.; Lopes, N.P.; de Oliveira, D.C.R. Post-Column sodiation to enhance the detection of polyacetylene glycosides in LC-DAD-MS analyses: An example from bidens gardneri (Asteraceae). Talanta, 2015, 135, 87-93.
Raina, R.; Etter, M. L. Liquid chromatography with post-column reagent addition of ammonia in methanol coupled to negative ion electrospray ionization tandem mass spectrometry for determination of phenoxyacid herbicides and their degradation products in surface water. Anal. Chem. Insights, 2010, 5ACI.S3148.
Carabias-Martínez, R.; Rodríguez-Gonzalo, E.; Revilla-Ruiz, P. Determination of weakly acidic endocrine-disrupting compounds by liquid chromatography-mass spectrometry with post-column base addition. J. Chromatogr. A, 2004, 1056, 131-138.
Cirigliano, A.M.; Cabrera, G.M. Differentiation of cyclosporin A from isocyclosporin A by liquid chromatography/electrospray ionization mass spectrometry with post-column addition of divalent metal salt. Rapid Commun. Mass Spectrom., 2014, 28(5), 465-470.
Cirigliano, A.M.; Rodriguez, M.A.; Gagliano, M.L.; Bertinetti, B.V.; Godeas, A.M.; Cabrera, G.M. Liquid chromatography coupled to different atmospheric pressure ionization sources-quadrupole-time-of-flight mass spectrometry and post-column addition of metal salt solutions as a powerful tool for the metabolic profiling of fusarium oxysporum. J. Chromatogr. A, 2016, 1439, 97-111.
Wang, S.; Cyronak, M.; Yang, E. Does a stable isotopically labeled internal standard always correct analyte response? J. Pharm. Biomed. Anal., 2007, 43(2), 701-707.
Rossmann, J.; Renner, L.D.; Oertel, R.; El-Armouche, A. Post-Column infusion of internal standard quantification for liquid chromatography-electrospray ionization-tandem mass spectrometry analysis - pharmaceuticals in urine as example approach. J. Chromatogr. A, 2018, 1535, 80-87.
Cheng, C.; Tsai, H-R. Analysis of steroids in yeast-mediated cell culture by on-line solid-phase extraction coupled high-performance liquid chromatography electrospray-ionization/mass spectrometry and novel continuous postcolumn infusion of internal standard technique. Anal. Chim. Acta, 2008, 623(2), 168-177.
Liao, H-W.; Tsai, I.L.; Chen, G-Y.; Lu, Y-S.; Lin, C-H.; Kuo, C-H. Quantification of target analytes in various biofluids using a postcolumn infused-internal standard method combined with matrix normalization factors in liquid chromatography-electrospray ionization mass spectrometry. J. Chromatogr. A, 2014, 1358, 85-92.
Liao, H-W.; Chen, G-Y.; Wu, M-S.; Liao, W-C.; Tsai, I-L.; Kuo, C-H. Quantification of endogenous metabolites by the postcolumn infused-internal standard method combined with matrix normalization factor in liquid chromatography-electrospray ionization tandem mass spectrometry. J. Chromatogr. A, 2015, 1375, 62-68.
Liao, H.W.; Chen, G.Y.; Wu, M.S.; Liao, W.C.; Lin, C.H.; Kuo, C.H. Development of a postcolumn infused-internal standard liquid chromatography mass spectrometry method for quantitative metabolomics studies. J. Proteome Res., 2017, 16(2), 1097-1104.
Liao, H.W.; Chen, G.Y.; Tsai, I.L.; Kuo, C.H. Using a postcolumn-infused internal standard for correcting the matrix effects of urine specimens in liquid chromatography-electro-spray ionization mass spectrometry. J. Chromatogr. A, 2014, 1327, 97-104.
González, O.; Van Vliet, M.; Damen, C.W.N.; Van Der Kloet, F.M.; Vreeken, R.J.; Hankemeier, T. Matrix effect compensation in small-molecule profiling for an LC-TOF platform using multicomponent postcolumn infusion. Anal. Chem., 2015, 87, 5921-5929.
Bonfiglio, R.; King, R.C.; Olah, T.V.; Merkle, K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun. Mass Spectrom., 1999, 13(12), 1175-1185.
Scott, E.P. Application of postcolumn ionization in the high-performance liquid chromatographic analysis of butabarbital sodium elixir. J. Pharm. Sci., 1983, 72(9), 1089-1091.
Kohler, M.; Leary, J.A. LC/MS/MS of carbohydrates with postcolumn addition of metal chlorides using triaxial electrospray probe. Anal. Chem., 1995, 67(19), 3501-3508.
Koch, W.; Forcisi, S.; Lehmann, R.; Schmitt-Kopplin, P. Sensitivity improvement in hydrophilic interaction chromato-graphy negative mode electrospray ionization mass spectrometry Using 2-(2-Methoxyethoxy)Ethanol as a post-column modifier for non-targeted metabolomics. J. Chromatogr. A, 2014, 1361, 209-216.
Mawhinney, D.B.; Stanelle, R.D.; Hamelin, E.I.; Kobelski, R.J. Enhancing the response of alkyl methylphosphonic acids in negative electrospray ionization liquid chromatography tandem mass spectrometry by post-column addition of organic solvents. J. Am. Soc. Mass Spectrom., 2007, 18(10), 1821-1826.
Elmongy, H.; Ahmed, H.; Wahbi, A.A.; Koyi, H.; Abdel-Rehim, M. Online Post-Column solvent assisted and direct solvent-assisted electrospray ionization for chiral analysis of propranolol enantiomers in plasma samples. J. Chromatogr. A, 2015, 1418, 110-118.
Feng, X.; Xiang, P.; Chen, H.; Shen, M. LC-MS-MS with post-column reagent addition for the determination of zolpidem and its metabolite zolpidem Phenyl-4-Carboxylic acid in oral fluid after a single dose. J. Anal. Toxicol., 2017, 41(9), 735-743.
Camenzuli, M.; Ritchie, H.J.; Dennis, G.R.; Shalliker, R.A. Reaction flow chromatography for rapid post column derivatisations: the analysis of antioxidants in natural products. J. Chromatogr. A, 2013, 1303, 62-65.

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Year: 2019
Page: [759 - 775]
Pages: 17
DOI: 10.2174/1573411015666190327170559
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