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Bio Analytical method development , validation & Transfer by using LC-MS/MS
Analytical Discussion
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About Authors:
Satya Lakshmi.B*, P.Raju vel, Dr. P. Venkateswara Rao, Sindhu.G, Nikil Kumar.K
A.M Reddy Memorial College of Pharmacy,
Narasaraopet, AN University, Guntur.

The reliability of quantitative assays in determination of drugs in biological fluids using High-performance liquid chromatography with Tandem Mass spectrometric determination (LC-MS/MS) detection methods and the integrity of resulting Pharmacokinetic data may not be absolute, in contrary to common perceptions and possible conjectures. The results may be adversely affected by lack of specificity and selectivity due to ion suppression caused by the sample matrix and interferences from metabolites. The advancements in the past few years and new technologies introduced can be used in enhancing LC-MS/MS Bio-analytical method development by reducing matrix effects. This Article reviews Automated Sample preparation and various extraction techniques like liquid-liquid extraction, Solid phase extraction and protein precipitation which plays an important role in sample preparation and detection by LC-MS/MS. Potential drawbacks during method development and validation are pointed out.

Reference Id: PHARMATUTOR-ART-1975

Method Development, Validation, Transfer is to understand Pharmacokinetics of drug and / or its metabolites in biological matrices. Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological matrix (plasma, urine, saliva, serum etc) play a significant role in evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. The rapid growth in the use of LC-MS/MS in recent years due to its advantages of high Sensitivity, Extreme Selectivity and increased rate of analysis.The other advantages of LCMS/MS include low detection limits, the ability to generate structural information, the requirement of minimal sample treatment and the possibility to cover a wide range of analytes differing in their polarities. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible for the intended use. The principle of MS is the production of ions from analyzed compounds that are separated or filtered on the basis of their mass-to charge ratio(m/z). Most of applications for quantitative bioanalysis use tandem mass spectrometers (MS/MS) that employs two mass analyzers – one for the precursor ion in the first quadrupole and the other for the product ion in the third quadrupole after the collision -activated dissociation of the precursor ion in a collision cell. The effective interface connection between LC  (operated under atmospheric pressure) and MS (operated under a high- vaccum environment) have made LC congenial with MS. Electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI), collectively called atmospheric pressure ionization (API), have matured into reliable interface necessary for quantitative LC-MS/MS bioanalysis. More recently, atmospheric pressure photo-ionization (APPI) also became an interesting alternative ionization source for quantitative LC-MS/MS. Successful use of LC-MS/MS requires understanding the mechanism of various sample extraction processes and the underlying principles of both chromatography and MS.

This article reviews Method development, various extraction techniques of sample preparations, Validation and Method transfer of robust LC-MS/MS with emphasis on important factors impacting the incurred sample analysis. We focused on the mature and established technologies used in most quantitative bioanalytical laboratories. The fundamental parameters for this validation include selectivity, Accuracy, Precision, Linearity and Range, Limit of detection, Limit of quantification, Recovery, Robustness and Stability.

Analytical method development is the process of creating a procedure to enable a compound of interest to be identified and quantified in a matrix. A compound can often be measured by several methods and the choice of analytical method involves many considerations, such as: chemical properties of the analyte, concentrations levels, sample matrix, cost of the analysis, speed of the analysis, quantitative or qualitative measurement, precision required and necessary equipment. The analytical chain describes the process of method development and includes sampling, sample preparation, separation, detection and evaluation of the results.

Traditional sequential method development requires sequential optimization of mass   spectrometric and chromatographic conditions, sample extraction, recovery of the analyte, and lack of interference and matrix effects for each method. This type of method development is timeconsuming, labor and instrument intensive and costly when several different LC-MS/MS methods for various types of analytes need to be developed. This approach has been identified as the major bottleneck for meeting the everincreasing needs for LC-MS/MS methods. In a Reearch paper ,”A concept of simultaneous development of multiple bioanalytical LCMS/MS methods” was presented. Optimal conditions of mass spectrometry, chromatography, and extraction were screened and developed for six structurally different analytes. Experimental designs for simultaneously determining and evaluating recovery, matrix effects, and chromatographic interference were proposed. In another presentation, processes were optimized so that a robust LCMS/ MS method was developed in a single working day .Two scientists work   simultaneously on the same project in a coordinated way: one focusing on sample preparation and other focusing on instrumentation. Important method parameters such as matrix suppression and recovery were investigated. Janiszewski et al. also proposed highthroughput method development approaches by simultaneous testing multiple SPE chemistries using a custom multiple sorbent 96-well plate with optimized extraction conditions for up to five analytes are determined in a single experiment.


The biological media that contain the analyte are usually blood, plasma, urine, serum etc. Blood is usually collected from human subjects by vein puncture with a hypodermic syringe up to 5 to 7 ml (depending on the assay sensitivity and the total number of samples taken for a study being performed). The venous blood is withdrawn into tubes with an anticoagulant, e.g. EDTA, heparin etc. Plasma is obtained by centrifugation at 4000 rpm for 15 min. About 30 to 50% of the original volume is collected Sadagopan et al. systematically investigated the feasibility of using EDTAanticoagulant in plasma to improve the throughput of LCMS/MS assays.

The purpose of sample preparation is to clean up the sample before analysis and/or to concentrate the sample. Material in biological samples that can interfere with analysis, the chromatographic column or the detector includes proteins, salts, endogenous macromolecules, small molecules and metabolic byproducts. A goal with the sample preparation is also to exchange the analyte from the biological matrix into a solvent suitable for injection into the chromatographic system. General procedures for sample preparation like liquid/liquid extraction, solid-phase extraction (SPE) and protein precipitation.

Liquid-Liquid extraction: It is based on the principles of differential solubility and partitioning equilibrium of analyte molecules between aqueous (the original sample) and the organic phases. Liquid-liquid extraction generally involves the extraction of a substance from one liquid phase to another liquid phase. Now-a-days traditional LLE has been replaced with advanced and improved techniques like liquid phase microextraction (LPME), single drop-liquid phase micro extraction (DLPME) and supported membrane extraction (SME).

Solid-phase extraction: SPE is a selective method for sample preparation where the analyte is bound onto a solid support, interferences are washed off and the analyte is selectively eluted. Due to many different choices of sorbents, SPE is a very powerful technique. SPE consists of four steps; conditioning, sample loading, washing and elution.

Column Solvation: The column is activated with an organic solvent that acts as a wetting agent on the packing material and solvates the functional groups of the sorbent. Water or aqueous buffer is added to activate the column for proper adsorption mechanisms.

Sample loading: After adjustment of pH, the sample is loaded on the column by gravity feed, pumping or aspirating by vacuum.

Column Washing: Interferences from the matrix are removed while retaining the analyte.

Target Compound Elution : Disruption of analyte-sorbent interaction by appropriate solvent, removing as little of the remaining interferences as possible

Fig 1: General Solid Phase Extraction Procedure.

Typically, sorbents used in SPE consists of 40 μm diameter silica gel with approximately 60 Aº pore diameters. To this silica gel, functional groups are chemically bonded, for different modes of action. The most commonly used format is a syringe barrel that contains a 20 μm frit at the bottom of the syringe with the sorbent material and another frit on top, referred to as packed columns. Extraction disks are also placed in syringe barrels. These disks consist of 8–12 μm particles of packing material imbedded into an inert matrix. Disks are conditioned and used in a similar way as packed columns. The major advantage of disks compared to packed columns is that higher flow rates can be applied. Analytes can be classified into four categories; basic, acid, neutral and amphoteric compounds. Amphoteric analytes have both basic and acidic functional groups and can therefore function as cations, anions or zwitterions, depending on pH.

Protein precipitation: Protein precipitation is often used in routine analysis to remove proteins. Precipitation can be induced by the addition of an organic modifier, a salt or by changing the pH which influence the solubility of the proteins. The samples are centrifuged and the supernatant can be injected into the LC system or be evaporated to dryness and thereafter dissolved in a suitable solvent. A concentration of the sample is then achieved. There are some benefits with the precipitation method as clean-up technique compared to SPE. It is less time consuming, smaller amounts of organic modifier or other solvents are used. But there are also disadvantages. The samples often contain protein residues and it is a non-selective sample cleanup method, there is a risk that endogenous compounds or other drugs may interfere in the LC-system. However the protein precipitation technique is often combined with SPE to produce clean extract.

Methanol is generally preferred solvent amongst the organic solvent as it can produce clear supernatant which is appropriate for direct injection into LC-MS/MS. Salts are other alternatives to acid and organic solvent precipitation. This technique is called as salt-induced precipitation. As the salt concentration of a solution is increased, proteins aggregate and precipitate from the solution.

One strategy for high-throughput bioanalytical analysis is to use well-established instrumentation; rigorous, standardized techniques; and automation, wherever possible, to replace manual tasks. Automation results in greater performance consistency over time and in more reliable methods transfer from site to site. Automated 96-well plate technology is well established and accepted and has been shown to effectively replace manual tasks. The 96-well instruments can execute automated off-line extraction and sample clean-ups. Automated solid-phase extraction (SPE), liquid/liquid extraction (LLE), and protein precipitation (PP) all can be performed in 96-well format. Both cartridge and disc in 96-well SPE formats have been successfully used. New trends in SPE have been reviewed by Poole. Polson et al. discussed optimization of protein precipitation based on effectiveness of protein removal and ionization effect.(Fig. 2) provides a general approach for automated 96-well sample analysis.In comparision to manual operation, automated sample preparation saved atleast 50% time.

Fig.2 :Automated 96- Well Sample analysis

With the initial sample clean-up using SPE, LLE or PP, unwanted compounds can be present still in higher concentrations than the analytes of interest. A second stage of clean-up, typically involving LC separation, further separates analytes of interest from the unwanted compounds.

Without this further separation, those unwanted and MS/MS unseen compounds present significant challenges. In the LCMS interface, these compounds compete with analytes for ionization and cause inconsistent matrix effects that are detrimental to quantitative LC-MS/MS performance

Reversed-phase LC has been traditionally used for the quantitative LC-MS/MS. Many drugs have basic functional groups, and acidic mobile phases are used and MS in the positive ion mode detects these compounds as the protonated ions. Ionization of polar compounds further decreases the analyte on-column retention on a reversed-phase column. To overcome this mismatch between reversed-phase LC and MS detection, other chromatographic materials were investigated to achieve better sensitivity and better on-column retention. Zirconia-based column may offer different retention mechanism from silica-based column and has been explored for quantitative bioanalytical LC-MS/MS application.

Once a Bioanalytical method is developed ,Various tests (collectively called Method Validation) are conducted to prove that the method can be used for its intended application. Typical parameters to validate are; include selectivity, accuracy, precision, linearity and range, limit of detection, limit of quantification, recovery, robustness and stability. General recommendation for analytical method validation, i.e. for pharmaceutical methods, can be found in The US Food and Drug Administration (FDA) guideline.

Selectivity exercise is carried out to assess the ability of the bioanalytical method to differentiate and quantify the analyte(s) in presence of other components in the sample. For selectivity, analyses of blank samples of appropriate biological matrix (plasma, urine, or other matrix) obtained from atleast six sources should be carried out. Each blank sample should be tested for interference and selectivity should be ensured at the lower limit of quantification (LLOQ).

The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%. The deviation of the mean from the true value serves as the measure of accuracy.

The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV. Precision is further subdivided into within-run, intra-batch precision or repeatability, which assesses precision during a single analytical run and between-run, interbatch precision or repeatability, which measures precision with time and may involve different analysts, equipment, reagents and laboratories.

Linearity and range:
A calibration curve is the relationship between instrument response and known concentration of the analyte. The calibration curve should be prepared in the same biological matrix as the samples and a calibration curve should be generated for each analyte. The range of the method is the concentration interval where accuracy, precision and linearity have been validated. The used calibration curve should be the simplest model that adequately describes the concentration-response relationship. The deviation should not exceed more than 20% from the nominal concentration of the LLOQ and not more than 15% from the other standards in the curve.

Limit of detection:
The limit of detection (LOD) is a characteristic for the limit test only. It is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated under the stated experimental conditions. The detection is usually expressed as the concentration of the analyte in the sample, for example, percentage, parts per million (ppm), or parts per billion (ppb).

Limit of quantification:
Lower limit of quantification: LLOQ is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. Detemining LLOQ on the basis of precision and accuracy is probably the most practical approach and defines the LLOQ as the lowest concentration of the sample that can still be quantified with acceptable precision and accuracy. LLOQ based on signal and noise ratio (s/n) can only be applied only when there is baseline noise, for example to chromatographic methods. Upper limit of quantification: ULOQ is the maximam analyte concentration of a sample that can be quantified, with acceptable precision and accuracy. The ULOQ is identical with the concentration of the highest calibration standards.

The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard. Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery.

Matrix effect:
Matrix effect is investigated to ensure that selectivity and precision are not compromised within the matrix screened. Three blank samples from each of at least six batches of matrix under screening are extracted. For matrix effect LQC (lower quality control), MQC (middle quality control) and HQC (higher quality control) spiking dilutions and internal standard dilution are spiked in the above extracted blank samples. Recovery comparison sample at LQC, MQC and HQC concentration level along with internal standard are prepared and screened.

According to ICH guidelines, The robustness of an analytical procedure is the measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness can be described as the ability to reproduce the (analytical) method in different laboratories or under different circumstances without the occurrence of unexpected differences in the obtained result(s), and a robustness test as an experimental set-up to evaluate the robustness of a method.

The stability of the analyte under various conditions should also be studied during method validation. The conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. The following stability conditions are required by FDA and are advisable to investigate;

Stock solution stability: The stability of the stock solution should be evaluated at room temperature for at least 6 hours.

Short-term temperature stability: The stability of the analyte in biological matrix at ambient temperature should be evaluated. Three aliquots of low and high concentration should be kept for at least 24 hours and then analysed.

Long-term temperature stability: The stability of the analyte in the matrix should exceed the time period from sample collection until the last day of analysis.

Freeze and thaw stability: The stability of the analyte should be determined, after three freeze and thaw cycles. Three aliquots of low and high concentration should be frozen for 24 hours and then thawed at ambient temperature.

Post-preparative stability: The stability of the analyte during stages of the analysis process should be evaluated.

Timely method transfer plays an important role in expediting drug candidates through development stages.Method transfer is not an easy task and requires careful planning and constant communication between the laboratory personnel involved in the transfer. Method transfer could occur within the same organization or between pharmaceutical companies and analytical service providers. To have a successful transfer, the bioanalytical method itself must be robust and the equipment differences between the delivering and receiving parties should be carefully evaluated. Use of standardized automation equipment has shown to be advantageous during the method transfer. Information on method transfer can be found in the literatures.

The method development and validation has an irreplaceable role in public health.Thus ,there is a need for LC-MS/MS practioners to consider few aspects, one should always ask the question “Whether the intended method can be used successfully for sample analysis”and should be in the position to assess the possibilities and act accordingly which will ensure a fruitful outcome having a considerable impact on the public health. This review article covers the recent advances in Bio-analytical LC-MS/MS methods.

1. Wells D.A, High throughput bioanalytical sample preparation: Methods and automation strategies, 1st ed. Amsterdam: Elsevier Science B. V. 2003.
2. Wells DA. High throughput bioanalytical sample preparation : methods and automation strategies, Progress in pharmaceutical and biomedical analysis, Amsterdam, London, Elsevier, xxx, 2003, 610.
3. Thurman E.M., Mills M.S., eds. Solid-phase extraction: Principles and practise. Chemical analysis: A series of monographs on analytical chemistry and its applications, Winefordner J.D, New York, John Wiley & sons Inc, 147, 1998.
4. Said R, Application of new technology LC-MS/MS for determination of therapeutic drugs, Doctoral degree thesis, Department of Medicine Division of Clinical Pharmacology Karolinska Institute, Stockholm, Sweden. 2010, 1-5.
5. Said R. Application of new technology LC-MS/MS for determination of therapeutic drugs, Doctoral degree thesis, Department of Medicine Division of Clinical Pharmacology Karolinska Institute, Stockholm, Sweden, 2010, 1-5.
6. U.S Department of Health and Human Services, Food and Drug Administration, Guidance for Industry, Bioanalytical Method Validation, May 2001.
7. Rosing H, Man WY, Doyle E, Bult A, Beijnen J H. Bioanalytical liquid chromatographic method validation- A review of current practices and procedures, J. Liq. Chrom. Rel. Technol, 23, 2000, 329-354.
8. Bressolle F, Bromet P, Audran M. Validation of liquid chromatography and gas chromatographic methods application to pharmacokinetics. J. Chromatogr. B. 686, 1996, 3-10.
9. Point. Journal of Pharmaceutical Sciences and Research. 3, 2009, 2-3.
10. Cappiello A, Famiglini G, Palma P, Pierini E, Termopoli V, Trufelli H. Overcoming matrix effects in liquid chromatography-mass spectrometry, Analytical Chemistry, 80, 2008, 9343- 9348.
11. Chiu ML, Lawi W, Snyder ST, Wong PK, Liao JC, Gau V. Matrix effects: A challenge toward automation of molecular analysis, Journal of the Association for Laboratory Automation, 15, 2010, 233-242.
12. Eric Reid, Ian D. Wilson. Methodological Survey in Biochemistry and Analysis: Analysis for Drug and Metabolites, Including Anti-infective Agents. 20, 1990, 1-57.
13. Food and Drug Administration Guidance for Industry Bioanalytical MethodValidation,2001.
14. Gad SC, Preclinical development handbook, New Jersey, John Wiley and Sons, 2008. Kazakevich Y, Lobrutto R, HPLC for Pharmaceutical Scientists, 1st ed; John Wiley & Sons, Inc.: New Jersey, 2007, 281-292.
15. Patel D. Matrix effect in a view of LC-MS/MS: an overview, International Journal of Pharmacy and Biological Sciences, 2, 2011, 559-564.
16. Rao R, kalakuntla K, Kumar S. BioanalyticalMethod Validation: A Quality Assurance Auditor View.
17. U.S Department of Health and Human Services, Food and Drug Administration, Guidance for Industry, Bioanalytical Method Validation, May 2001.
18. Hsieh,Y.; Merkle,K,; Wang,G.; Brisson, J-M.; Korfmacher, W.A.Anal.Chem.,2003,75,3122.
19. Yang,C.;Henion,J.J.Chromatogr.A,2002,970,155.
20. Brewer,E.;Heenion,J.J.Pharm.sci.,1998,87,395.
21. Lee,M.S.;Kerns,E.D.Mass Spectrom.Rev., 1999,18,187.
22. Jemal,M.Biomed.Chromatogr.,2000,14,351.
23. Brockman, A.H.;Hiller, D.L.; Cole, R.O.Curr.Opin. Drug Disc.Dev.,2000,3,432.
24. oliveria, E.J.;Watson,D.G.Biomed.Chromatogr.,2000,14,351.
25. Law,B.;Temesi,D.J. Cjromatogr., B 2000,748,21.
26. Plumb R.; Dear , G.J.;Mallett,D.N.;Higton , D.M.;Pleasance ,S.; Biddlecombe , R.A.Xenobiotica,2001,31,599.
27. Shou,W.Z.;Jiang,X.;Beato ,B.D.;Naidong,W.Rapid Commun. Mass Spectrom., 2001, 15,466
28. Matuszewski, B.K.; Constanzer, M.L.; Chavez-Eng, C.M.Anal.Chem., 1998,70,882.
29. Weng,N.;Halls, T.D.J. Pharmaceutical Technology,2002,26,102.
30. Hsieh,Y.; Merkle, K.;Wang, G.Rapid Commun. mass Spectrom.,2003,17,1775.
31. Naidong,W.;Bu,H.;Chen,Y-L.;Shou, W.Z.; Jiang, X.; Halls, T.D.J.J.PharmBiomed. Anal.,2002,28,1115.
32. Pan,J.;Junga,H.;Sun,H.; Jiang,X.; Weng, N.51th Annual ASMS Conference, Montreal, Quebec, Canada , June 8-12,2003.
33. Allanson,J.P.; Biddlecombe, R.A.; Jones, A.E.; Pleasance, S.Rapid Commun. Mass Spectrom.,1996,10,811.
34.Rose,M.j.; Merschman, S.A.;Eisenhandler,R.;Woolf,E.J.; Yeh,K.C.; Lin,L.; Fang,W.; Hsieh,J.; Braun,M.P.; Gatto,G.j.; Matuszewski, B.K.J.Pharm. Biomed.Anal.,2000,24,291.
35.Eerkes,A.;Addison,T.; Jiang,X.; Naidong,W.J. Chromatogr.B, 2002,768,277.
36. Steinborner S.; Henion,J.Anal.Chem.,1999,71,2340.
37. Jemal, M.; Teitz, D.; Ouyang, Z.; Khan, S. J. Chromatogr. B, 1999,732, 501.
38. Bolden, R.D.; Hoke II, S.H.; Eichold, T.H.; McCauley-Myers,D.L.; Wehmeyer, K.R. J. Chromatogr. A, 2002, 772, 1.
39. Ramos, L.; Bakhtiar, R.; Tse, F.L.S. Rapid Commun. Mass Spectrom., 2000, 14, 740.
40. Shen, Z.; Wang, S.; Bakhtiar, R. Rapid Commun. Mass Spectrom.,2002, 16, 332.
41. Eerkes, A.; Shou, W.Z.; Naidong. W. J. Pharm. Biomed. Anal.,2003, 31, 917.
42. Ji, Q.C.; Reimer, M.T.; El-Shourbagy, T.A. J. Chromatogr. B,2004, 805, 67.
43. Watt, A.P.; Morrison, D.; Locker, K.L.; Evans, D.G. Anal. Chem.,2000, 72, 979.
44. Chen, Y-L.; Felder, L.; Jiang, X,; Naidong, W. J. Chromatogr. B, 2002, 774, 67.
45. Gritti, F.; Piatkowski, W.; Guiochon, G. J. Chromatogr. A, 1999,832, 51.
46. Bidlingmaier, B.; Unger, K.K.; von Doehren, N. J. Chromatogr. A,1999, 832, 11.
47. Lubda, D.; Cabrera, K.; Kraas, W.; Schaefer, C.; Cunningham, D.LCGC, 2001, 19, 1186.
48. Wu, J.T.; Zeng, H.; Deng, Y.; Unger, S.E. Rapid Commun. Mass Spectrom., 2001, 15, 1113.
49. Hsieh, Y.; Wang, G.; Wang, Y.; Chackalamannil, S.; Brisson, J.M.; Ng, K.; Korfmacher, W.A. Rapid Commun. Mass Spectrom.,2002, 16, 944.
50. Deng, Y.; Wu, J.T.; Lloyd, T.L.; Chi, C.L.; Olah, T.V.; Unger, S.E.Rapid Commun. Mass Spectrom., 2002, 16, 1116.
51. Dear, G.; Mallett, D.N.; Higton, D.M.; Roberts, A.D.; Bird, S.A.;Young, H.; Plumb, R.S.; Ismail, I.M. Chromatographia, 2002, 55,177.
52. Chen, Y-L.; Junga, H.; Jiang, X.; Naidong, W. J. Sep. Sci., 2003,26, 1509.
53. Hsieh, Y.; Merkle, K.; Wang, G. Rapid Commun. Mass Spectrom.,2003, 17, 1775.
54. Mao, D.; Zhou, S.; Weng, N.; Jiang, X. Proceedings of the 52nd ASMS Conference on Mass Spectrometry and allied Topics;Nashville, Tennessee, 23-27 May 2004.
55. Naidong, W.; Shou, W.Z.; Chen, Y-L.; Jiang, X. J. Chromatogr. B,2001, 754, 387.
56. Stokes, P.; O’Connor, G. J. Chromatogr. B, 2003, 794, 125.
57. Jemal, M.; Xia, Y.Q. Rapid Commun. Mass Spectrom., 1999, 14,422.
58. Veuthey, J-L.; Souverain, S.; Rudaz, S. Ther. Drug Monit., 2004,26, 161.
59. Ke, J.; Yancey, M.; Zhang, S.; Lowes, S.; Henion, J. J.Chromatogr. B, 2000, 742, 369.
60. Parise, R.A.; Ramanathan, R.K.; Zamboni, W.C.; Egorin, M.J. J.Chromatogr. B, 2003, 783, 231.
61. Croubels, S.; de Baere, S.; de Backer, P. J. Chromatogr. B, 2003,788, 167.
62. Lakso, H.; Norström, A. J. Chromatogr. B, 2003, 794, 57.
63. Keevil, B.G.; Lockhart, S.J.; Cooper, D.P. J. Chromatogr. B, 2003,794, 329.
64. Stokes, P.; O’Connor, G. J. Chromatogr. B, 2003, 794, 125.
65. Rule, G. and Henion, J. Am. Soc. Mass Spectrom., 1999, 10, 1322.
66. Needham, S.R.; Ye, B.; Smith, R.; Korte, W.D. J. Chromatogr. B, 2003, 796, 347.
67. Frerichs, V.A.; Tornatore, K.M. J. Chromatogr. B, 2004, 802, 329.

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