Bioanalytical Method Validation A Comprehensive Review

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Int J Pharm Sci Rev Res 56 1 May June 2019 Article No 09 Pages 50 58 ISSN 0976 044X. Requirements 1 Authenticated source for Biological OPTIMIZATION OF MOBILE PHASE pH. Matrix 2 Reference or working Standards 3 Solvents. and Chemicals 4 Chromatographic Devices Instruments. Columns 5 Well trained Man Power 6 Literature,Method Development Steps. 1 Literature search for drugs,2 Physicochemical properties of the. 3 Dose Cmax of the compound,4 Selection of chromatographic device. Figure 1 It is the representation of graph between. 5 Reference standard preparation, Retention vs PH for a hypothetical acid a and base b. 6 Selection of Internal Standard, 7 Tuning of the compound OPTIMIZATION OF MOBILE PHASE pH.
8 Optimization of chromatographic,parameters,9 Optimization of Extraction procedure. 10 Sample storage,1 Physicochemical properties of the compound. a Solubility b pKa C molecular weight d molarity e pH. 2 Dose Cmax of the compound, a These two are required to find out the required LLOQ. level and to fix the required Linearity range Figure 2 Representation of graph between Time min vs. b As per regulatory guidelines Basic analytes, c LLOQ should be 5 half life of the Cmax The effect of small changes in mobile phase pH on. separation,d ULOQ should be 2 2 5 times of the Cmax.
a Basic analytes p anisidine m toluidine 4, 3 Selection of chromatographic device chloroaniline 3 aminobenzonitrile in retention order. a Selection of Chromatographic device is depends on the 27 73 methanol phosphate buffer. required sensitivity 7 Optimization of Extraction procedure. b Sensitivity ranged from sub pg mL to g mL level Types of Extractions. 4 Selection of Internal Standard 1 Protein Precipitation PPT 2 Liquid Liquid Extraction. Internal standard should preferably labeled compound if LLE 3 Solid Phase Extraction SPE 4 Hybrid Extraction. not Structurally similar or pKa similar 1 LIQUID LIQUID EXTRACTION. 5 Tuning of the compound Liquid liquid extraction is the direct extraction of the. a Source dependent Parameters Curtain gas nebulizing biological material with a water immiscible solvent An. gas Sheet gas Source Voltage etc aqueous sample e g plasma urine and an. immiscible organic solvent are mixed to remove the. b Compound Dependent Parameter Declustering analyte into the organic phase for injection into an. Potential entrance potential collision Energy Exit analytical system. Potential etc,Provide good recovery and clean sample. 6 Optimization of chromatographic parameters,Used for the extraction of basic and acidic drugs. Mobile Phase Mobile Phase Ratio Column Flow from biological samples. rate Temperature Injection volume Carry Over,An efficient method especially to eliminate salts. Time consuming Not suitable for extraction of, several analytes with different polarity Evaporation.
step is often required prior to analysis, The analyte is isolated by partitiong between the organic. phase and the aqueous phase The distribution ratio is. International Journal of Pharmaceutical Sciences Review and Research. Available online at www globalresearchonline net 51. Copyright protected Unauthorised republication reproduction distribution dissemination and copying of this document in whole or in part is strictly prohibited. Int J Pharm Sci Rev Res 56 1 May June 2019 Article No 09 Pages 50 58 ISSN 0976 044X. effected by a number of factors Choice of extracting Table 1 Comparison of Sample Techniques. solvent PH of aquephase relative lipophilicity or,Parameter PPT LLE SPE. hydrophobicity of the analyte ex teritary butylmethyl. ether dichloro methane hexane diethyl ether etc Workability Less More More. Solid Phase Extraction SPE Selectivity Bad Good Very good. Solid phase extraction is carried out by using a six step High Low Low. suppression,Automation Low Low High, Condition and equilibrium sample pre treatment sample Analyte Hydrophilic. loading washing drying elution Highly efficient Cost Hydrophilic Lipophilic. suitability Lipophilic, effective High reproducibility Advantageous such as. Cost Low High High, separation and concentrating of trace analytes Nature.
and amount of the sorbent Loaded sample volume Hybrid extraction Technique. Enough recovery Composition and volume of the, washing and elution solutions are effective a sorbent of Selective extraction of Analyte by using the combination. 50 200 mg is used as cartridge to separate the required of two or more extraction techniques E g PPT and SPE or. analytes from a complex matrix PPT and LLE Hybrid extraction is intended for Improve. Specificity Improve detection limits Improve recovery. 1 Reverse phase SPE,Method Validation,2 Normal phase SPE. 3 Ion exchange SPE Method validation is a process to demonstrate that a. method will successfully meet or exceed the minimum. REVERSE PHASE SPE standards recommended in the guidelines. Polar sample matrix includes nonpolar analytes using Validation involves documenting through the use of. nonpolar sorbent specific laboratory investigations that the performance. Sorbents used Bonded silica C4 C8 C18 and Phenyl with characteristics of the method are suitable and reliable for. 40 m particle size and 60 pore size and polymer the intended analytical applications The acceptability of. sorbent as polystyrene Retention mechanism is based on analytical data corresponds directly to the criteria used to. Hydrophobic interactions between analytes and validate the method. nonpolar sorbent materials less selective polar sample Types of method validation. matrix includes nonpolar analytes using nonpolar sorbent. 1 Full Validation,NORMAL PHASE SPE,2 Partial validation. Polar analytes in nonpolar matrices using polar sorbents. 3 Cross validation, Sorbents used Silica with polar functional groups Si CN. Si NH2 Si Diol and pure silica Retention mechanism is Full Validation. based on hydrogen bonding between analytes and Full validation is important when developing and. sorbent implementing a Bioanalytical method for the first. ION EXCHANGE SPE time, Full validation is important for a new drug entity.
Most selective method for charged analytes ANIONIC A full validation of the revised assay is important if. ANALYTES ACIDIC DRUGS Isolated with metabolites are added to an existing assay for. quaternary amine bonded silica or Si NH2 as anion quantification. exchange CATIONIC ANALYTES BASIC DRUGS Si Strong, cation exchange propyl sulfonic acid bonded Si weak Parameters to be Validate 23 28. cation exchange Carboxy propyl phase 1 Specificity 2 Auto sampler Carry Ove 3 Sensitivity 4. PROTEIN PRECIPITATION PPT Precision Accuracy 5 Recovery 6 Matrix Factor 7. Ruggedness 8 Stabilities 9 linearity, Appropriate for plasma or blood samples especially at. high analyte concentration Induced by the addition of A SPECIFICITY SELECTIVITY. miscible organic solvent acetonitrile acetone or Specificity is for identification of analyte s or metabolite. methanol Salt aluminium chloride Metal ions zinc s or matrix components interferences at their respective. sulphate Changing the sample pH to alter the nature of RT s on Biological Matrix Selectivity is evaluated by. the solution Trichloro acetic acid Perchloric acid Meta injecting extracted blank plasma and comparing with the. phosphoric acid and Tungstic acid Different sample response of extracted LLOQ samples processed with. techniques are shown in the Table 1 internal standard. International Journal of Pharmaceutical Sciences Review and Research. Available online at www globalresearchonline net 52. Copyright protected Unauthorised republication reproduction distribution dissemination and copying of this document in whole or in part is strictly prohibited. Int J Pharm Sci Rev Res 56 1 May June 2019 Article No 09 Pages 50 58 ISSN 0976 044X. Procedure MQC Middle of the Calibration curve, 6 Lots of intended anti coagulant 2 Lots of Heamolized 2 LQC Less than 3 time of the LLOQ. Lots of Lipemic, Acceptance criteria Accuracy should be within 85 to. Acceptance criteria 115 except LLOQ for LLOQ it should be 80 to 120. CV should be 15 except LLOQ for LLOQ it should be, Should less than 20 of the LLOQ response for analyte.
s Should be less than 5 of the internal standard,response Recovery. Auto Sampler Carry Over ASCO Determination of Extraction efficiency The detector. response obtained from an amount of the analyte added. Procedure Diluent ULOQ Diluent LLOQ, to and extracted from the biological matrix compared to. Acceptance criteria Response of the diluent injected the detector response obtained for the true. after ULOQ should be less than 5 of the LLOQ concentration of the pure authentic standard Recovery. pertains to the extraction efficiency of an analytical. Sensitivity,method within the limits of variability. Sensitivity is defined as the lowest analyte concentration. in the matrix that can be measured with acceptable. accuracy and precision i e LLOQ The lower limit of AQ Injections six replicates of 3 different levels H M L. quantification LLOQ is the lowest concentration of Post Extracted Injection six replicates of 3 different. analyte in a sample which can be quantified reliably with levels H M L. an acceptable accuracy and precision The LLOQ is,Acceptance criteria. considered being the lowest calibration standard see. Accuracy and Precision In addition the analyte signal of CV of Mean Recovery from three different. the LLOQ sample should be at least 5 times the signal of a concentrations should be within 15. blank sample The LLOQ should be adapted to expected. Matrix Effect or Matrix Factor or ISTD Normalization. concentrations and to the aim of the study As an, example for bioequivalence studies the LLOQ should be Determination of Matrix ions effect on Analyte or internal.
not higher than 5 of the Cmax while such a low LLOQ standard Matrix effect studied by comparing the response. may be not necessary for exploratory pharmacokinetic of extracted samples spiked before extraction with. studies response of the blank matrix sample to which analyte has. been added at the same nominal concentration just,before injection. Injecting the 6 replicates of LLOQ concentration, Procedure AQ Injections six replicates of 3 different. Acceptance criteria levels H M L Post Extracted Injection six replicates of. 3 different levels H M L with 6 different lots of Blank. Accuracy should be within 80 to 120 CV should be, Acceptance criteria ISTD normalization should be within. Precision Accuracy P A,Ruggedness, The precision is the closeness of agreement i e degree. Different Analyst, of scatter among a series of measurements obtained.
from multiple sampling of the same homogenous sample Different Column. under the prescribed conditions The acceptance criteria. Different Instrument,is 15 CV At LOQ 20 deviation is acceptable. One Precision Accuracy for each change,Closeness of determined value to the true value. Closeness to the nominal Value The mean value should be Acceptance criteria. within 15 of the theoretical value except at LLOQ,Accuracy should be within 85 to 115 except LLOQ. where it should not deviate by more than 20,for LLOQ it should be 80 to 120. CV should be 15 except LLOQ for LLOQ it should, Injecting 6 replicates of at least 3 different levels of the be 20.
concentrations,HQC Near to the ULOQ, International Journal of Pharmaceutical Sciences Review and Research. Available online at www globalresearchonline net 53. Copyright protected Unauthorised republication reproduction distribution dissemination and copying of this document in whole or in part is strictly prohibited. Int J Pharm Sci Rev Res 56 1 May June 2019 Article No 09 Pages 50 58 ISSN 0976 044X. Stability also fails then the method should be revised before. restarting validation Although the calibration curve. Chemical stability of an analyte in a given matrix under. should preferably be prepared using freshly spiked. specific conditions for given time intervals Analyte. samples it is allowed to use previously prepared and. change in any respect affect the chromatographic, stored calibration samples if supported by appropriate. behavior which may complicate the method,stability data. development,Stability in Matrix,Calibration curv,Bench Top Stability BT Freeze Thaw Stability FT. The response of the instrument with regard to the, Long Term Stability LT Wet Extract Stability WE Auto.
concentration of analyte should be known and should be. sampler Stability ASS Blood Stability BS Analyte and. evaluated over a specified concentration range The. IS stock stability in solvent Short Term Stability in matrix. calibration standards should be prepared in the same. In injector stability in matrix, matrix as the matrix of the intended study samples by. spiking the blank matrix with known concentrations of Stability in Aqueous Solution. the analyte There should be one calibration curve for. Long term Short term Stock Solution Stability, each analyte studied in the method validation and for. each analytical run Ideally before carrying out the Long term short term working solution stability. validation of the analytical method it should be known. Acceptance Criteria, what concentration range is expected This range should. be covered by the calibration curve range defined by the For matrix stabilities Mean stability should be with 85 to. Keywords Bioanalytical method development validation parameters documentation application biological matrices pharmacokinetic studies INTRODUCTION io analysis1 is defined as Quantification of analyte metabolites in human biological matrix Blood Plasma Serum Urine faeces skin saliva and other organ tissues by using chromatographic2 10 devices eg HPLC LC MS GC MS etc Bio

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