Liquid chromatography—mass validation

For method tryout, run a control sample and two fortifications from each site. One fortification should be done at the LOQ and the other at the highest expected residue level, perhaps 1000 x LOQ. If the recoveries are within the acceptable range of 70-120% and there are no interferences, proceed with the method validation. If interferences are present which prevent quantitation of the analyte, try additional cleanup steps with SPE or use a more selective detection method such as liquid chromatography/mass spectrometry (LC/MS). 

High performance liquid chromatography-mass spectrometric methods Nitin et al. [75] developed and validated a sensitive and selective liquid chromatography-tandem mass spectrometric method (LC MS MS) for the simultaneous estimation of bulaquine and its metabolites primaquine in monkey plasma. The mobile phase consisted of acetonitrile ammonium acetate buffer (20 mM, pH 6) (50 50, v/v) at a flow rate of 1 mL/min. The chromatographic separations were achieved on two Spheri cyano columns (5 pm, 30 cm x 4.6 mm), connected in... 

Many of the more established techniques have been validated through collaborative studies which becomes of greater importance as laboratories seek to become accredited via ISO, EN or related systems where the use of official or well validated methods is mandatory. New instrumental techniques are constantly being reported in the literature but it often requires many years before procedures are introduced, validated and then applied within the food industry. Recent techniques that can be included in this category are capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS). In time procedures based on these techniques will also become accepted as routine methods and are likely to be adopted by some of the official international bodies like the AOAC International, CEN, ISO, etc. 

Kratzsch C, Peters FT, Kraemer T, Weber AA, Maurer HH. 2003. Screening, library-assisted identification and validated quantification of fifteen neuroleptics and three of their metabolites in plasma by liquid chromatography/ mass spectrometry with atmospheric pressure chemical ionization. J Mass Spectrom 38 283. 

Hayward, M. J., Hargiss, L. O., Munson, J. L., Mandiyan, S. P. and Wennogle, L. P., Validation of Solubility Measurements Using Ultra-Filtration Liquid Chromatography Mass Spectrometry (UP-LC/MS), American Society for Mass Spectrometry 2000 Conference Abstract, Long Beach, CA, USA, 2000. 

Mohn T, Cutting B, Ernst B, Hamburger M (2007) Extraction and analysis of intact glucosinolates-a validated pressurized liquid extraction/liquid chromatography-mass spectrometry protocol for Isatis tinctoria, and qualitative analysis of other cruciferous plants. J Chromatogr 1166 142-151... 

Chi C, Liang L, Padovani P et al. (2003) Development and validation of a liquid chromatography-mass spectrometric method for the determination of DPC 423, an antithrombotic agent, in rat and dog plasma. J Chromatogr B Analyt Technol Biomed Life Sci 783( 1) 163—172 Zimmer D, Muschalek V, Muller C (2000) Determination of met-rifonate enantiomers in blood and brain samples using liquid chromatography on a chiral stationary phase coupled to tandem mass spectrometry. Rapid Commun Mass Spec-trom 14(15) 1425-1432... 

Maurer, H.H., Tenberken, O., Kratzsch, C., Weber, A., Peters, F.T. Screening for library-assisted identification and fully validated quantification of 22 beta-blockers in blood plasma by liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization. J. Chromatogr. A 1058, 169-181 (2004)... 

Most of these measurements are still routinely perfonned with immunoassays because of their simplicity, rapidity and relatively low cost, and despite occasional concerns about their reliability and validity. Serum total T has also been quantitated by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and LC-MS-MS. MS-based methods are often used in research smdies or to confirm immunoassay results. The advantage of chromatography coupled with MS is the high specificity not available with immunoassays because they are susceptible to cross-reactions. An immunoassay might measure a host of structurally related compounds in addition to the target analyte. 

Cross-validation is conducted when two validated bioanalytical methods are used within the same study or submission, for example, ELISA assay to Biacore and ELISA to a liquid chromatography/mass spectrometry. It is recommended that test samples (spiked and/or pooled incurred samples) be used to cross-validate the bioanalytical methods. Data should be evaluated using an appropriate predefined acceptance criteria or statistical method [12,13]. It should be cautioned that many times the methods that are being cross-validated may not have the same range of quantification. In these situations, it is necessary to prepare spiked samples within the range that are common to both methods for comparison. 

Teh CH, Murugaiyah V, (Than KL (2011) Developing a validated liquid chromatography-mass spectrometric method for the simultaneous analysis of five bioactive quassinoid markers for the standardization of manufactured batches of Eurycoma longifolia Jack extract as antimalarial medicaments. J Chromatogr A 1218 1861-1877... 

Teixeira H, Verstraete A, Proen a P, Corte-Real F, Monsanto P, Vieira DN (2007) Validated method for the simultaneous determination of Delta9-THC and Delta9-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography-mass spectrometry with electrospray ionization. Forensic Sci Int 170(2-3) 148-155... 

Pichini, S., Pacifici, R., Pellegrini, M., Marchei, E., Lozano, J., Murillo, J., VaU, O. and Garcia-Algar, O., Development and validation of a high-performance liquid chromatography— Mass spectrometry assay for determination of amphetamine, methamphetamine, and methyl-enedioxy derivatives in meconium. Anal. Chem., 76, 2124-2132 (2004). 

Rudaz, S., S. Souverain, C. Schelling et al. 2003. Development and validation of a heart-cutting liquid chromatography-mass spectrometry method for the determination of process-related substances in cetirizine tablets. Anal. Chim. Acta. 492 271-282. 

Full acceptance of HPLC/MS methods by the US EPA OPP as enforcement methods occurred between 1998 and 2001. For example, in 1998, the EPA OPP accepted HPLC/MS (without MS/MS) methods as primary enforcement methods, and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) only was suitable for confirmatory methods. However, in 2001, HPLC/MS/MS methods also became acceptable for primary enforcement. Table 4 summarizes the types of methods that were validated by the EPA OPP method validation program, for both food tolerance enforcement methods and environmental chemistry methods. 

Wille K, Vanden Bussche J, Noppe H, De Wulf E, Van Caeter P, Janssen CR, De Brabander HF, Vanhaecke L (2010) A validated analytical method for the determination of perfluorinated compounds in surface-, sea- and sewagewater using liquid chromatography coupled to time-of-flight mass spectrometry. J Chromatogr A 1217(43) 6616-6622... 

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