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Dushyant Patel LC-MS/MS Method

S.K.P.C.P.E.R., Kherva 41 M .Pharm. Thesis

plasma where 25 µl of diluent was added.

b) Plasma sample (100 µl) (blank plasma, zero standard, calibration standard, quality

control samples) and study samples were added in micro tube using a micro pipette.

c) Water (100 µl) was added and vortexed to mix. The above plasma mixture was loaded on

previously conditioned (conditioned with 1 ml of methanol and then with 1 ml of water)

Orochem SPE columns (Orpheus, 30 mg, 1 ml, DVB-LP). The cartridges were washed

with 1 ml of water and eluted with 1ml of elution solution & vortexed it.

d) Samples (5.0 µl) prepared as above was injected.

4.2.13 METHOD VALIDATION OF LC-MS/MS METHOD:

Pre method validation:

1. Pre method validation is a generic that provides concretes, statistical evidence for

acceptable method performance prior to initiating a bioanalytical method validation.

2. A large number of replicate spiked matrix samples were analyzed in a single batch

before method validation.

3. Pre method validation experiment was performed for system suitability, specificity &

selectivity, sensitivity, three linearity, three between-batches or inter-batch

accuracy and precision, recovery and carry over effect check.

4. All pre method validation samples prepared according to describe in sample preparation

section.

5. The utility of this pre method validation approach was illustrated using actual laboratory

data. The process of interpreting the results and drawing conclusions about assay

viability was demonstrated.

6. The resulting conclusions provide sufficient background information to indicate if an

assay procedure can be ready to enter in the validation process.

Method validation and characteristics of the Method:

A. Chromatography:

¾ Representative chromatograms of blank plasma, zero standard, LLOQ, ULOQ, LQC,

MQC, and HQC samples GBP are represented in Figure No 4.6, 4.7, 4.8, 4.9, 4.10, 4.11

and 4.12, respectively.

B. Specificity and selectivity:

¾ Obtain samples of the relevant biological matrix (e.g., plasma, serum, blood, etc.)

collected under controlled conditions.

¾ For plasma matrix, 08 normal plasma lots (04 different plasma lots with the

anticoagulant to be used during method validation and 04 different plasma lots with the

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