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To ensure the mass spectrum for a particular Analyte/Metabolite tuning can performed .To achieve the maximum mass spectral sensitivity tuning is an essential process, which involves optimising the voltages (capillary, cone, extractor and RF lens voltages), currents, ion source and flow parameters. Perflourotributylamine (PFTBA) is used as a standard to tune the mass spectrometer.
Tuning of mass spectrometer provides the information regarding:
Ion sources parameters (no of ions produced and no ions directed towards the mass filter).
Mass filter parameters (sensitivity, mass resolution, peak widths and mass assignments).
Detector parameters(detector sensitivity ,magnitude of the signal)
Optimisation of mass parameters:
Optimisation of mass parameters involves setting of proper mass range, proper threshold, and verification of system performance and maintaining of system performance. In order to get the proper mass spectral resolution threshold setting is done which involves adjusting the capillary voltage, cone voltage, extractor voltage and RF lens voltage. Verification of system performance ensures system sensitivity, chromatographic performance and back ground signal. Maintenance of system performance ensures temperature in MS unit, continuous carrier-gas flow into the MS and maintenance of vacuum.
Table1.2A: Tuning parameters
LM Resolution 1
HM Resolution 1
Ion Energy 1
RF Lens (V)
Source Temp (0 C)
Desolvation Temp (0 C)
Cone Flow (L/h)
LM Resolution 2
Desolvation Flow (L/h)
HM Resolution 2
Collision gas Pressure
Ion Energy 2
Optimisation of chromatographic conditions:
Selection of column
Selection mobile phase
Optimisation of mobile phase composition
Column oven temperature
Auto sample temperature
Optimisation of Extraction procedure (sample preparation):
Objective of the sample preparation is removal of interfering compounds, Extraction of sample in to a suitable solvent and pre -concentration of the sample. Sample preparation improves specificity, reproducibility, accuracy, precision, recovery and stability of the sample and instrument life during sample analysis.
Sample preparation method:
Protein precipitation method
Liquid -liquid Extraction method
Solid-Phase Extraction procedure method
Hybrid Extraction method
ï¶ Protein precipitation: Involves denaturation of proteins by using water-miscible organic solvents (methanol, ACN, ethanol, etc.) and acids. Denaturation of proteins involves by changing the pH of the sample, addition of organic solvent and increase the salt concentration of sample.
Procedure: I part of the sample can be diluted with a two-three parts of precipitating agent, then samples were vertexed and fallowed by addition of extraction solvent. Then the samples were centrifuged at high rpm fallows the collection of supernatant liquid and the sample were directly analysed. If required concentrated samples, the supernatant sample were evaporated to dryness and reconstituted before analysis.
Advantages: Simple, inexpensive, universal method for sample extraction procedure.
Disadvantages: matrix components cannot be separated efficiently and it will decrease the efficiency of Ionization process, analytical column and instrument life, and affects the sample recovery, accuracy, linearity and specificity.
ï¶Liquid-liquid extraction: It involves by partitioning of sample (matrix) between two immiscible solvents (i.e., organic and aqueous phase).Liquid-liquid extraction mainly based on the solubility (partition coefficient) of the sample between the two phases.
Procedure: Mobile phase and organic solvent were added to the biological matrix and vertexed fallowed by addition of extraction solvent. The samples were centrifuged at specified rpm, the supernatant liquid was collected. The extracted samples were directly analysed. If required concentrated samples, the supernatant samples were evaporated to dryness and reconstituted before analysis. Commonly used extraction solvents are (t-BMA, n-hexane, dichloro methane).It is inexpensive method compare with SPE; it can efficiently extracts the samples and decreases the analytical problems during analysis.
ï¶Solid-phase extraction: It involves the adsorption of the targeted analyte on the solid phase support. By using suitable organic solvents (methanol, ACN, t-BMA, etc.) the targeted analyte can be collected.
Basic steps involved the solid phase extraction:
1. Conditioning: The SPE cartridges to be conditioned by using dilute organic solvents (methanol, ACN, etc.).
The main purpose of conditioning is as fallows
Avoid excessive drying of stationary phase bed
To activate the sites of stationary phase and removes dust, moisture from the stationary phase.
2. Sample-pre treatment: It involves the addition of recommended amount of mobile phase, ISTD and organic solvent to the sample and addition of suitable buffering agent to the sample.
3. Sample application: From the top the SPE cartridges at a slow rate the sample to be applied, the vacuum pump place a role and collect the matrix from the cartridges. The targeted analyte will bounded to the Stationary phase itself.
4. Washing/rinsing of stationary bed: This is mainly for the removal of interferences and matrix components from the cartridges by using solvents (water, buffers, and very dilute organic solvents).
5. Drying: It can be done by applying vacuum for recommended time(2-3min).it is mainly for the removal of excess washing solvents, avoid air bubble formation which leads to blockage of cartridges.
6. Elution: Can be performed by passing elution solvents (methanol, ACN, t-BMA, dichloromethane, etc.) from the cartridges the sample. Here organic solvent place a role to weaken the bonds between the Analyte to the sorbent. In each step applying recommended vacuum place a key role during extraction.
ï³ Buffering agent:
Buffering agent selection mainly based on pKa of drug. If the pH of the buffer 1.5 units above its pKa value, the analyte will ionized and selects aqueous phase, only less polar interferences are goes to organic solvents. If the pH drugs below its pKa, the analyte will unionized and extracted in to the organic phase by leaving most polar interferences in the aqueous phase. So the buffering agent mainly used to maintain the pH of the Analyte.
ï³ Mobile phase buffering agent:
For sample analysis buffer pH should be selected as Â±2 of its pKa value. Some times higher buffer concentration may affect the instrument parts. Mostly used Buffers are ammonium formate, ammonium acetate, etc. They will maintain the sample pH and also increases the extraction efficiency, sensitivity, linearity of the sample during analysis.
1.3 BIOANALYTICAL METHOD VALIDATION
Method validation can be defined as according to ICHQ.2B guidelines "Establishing documented evidence, which provides high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics".
Method validation can be performed after the method development and it gives documented information regarding the linearity, accuracy, specificity and stability parameters of the analyte. And it also demonstrates the specific method is applicable for the Quantitation of analyte in the biological matrices which are reproducible for intended and long term use.
Validation parameters have been proved by considering the sample preparation, sample extraction procedure, chromatographic parameters. If the method proving all the validation parameters are within the acceptance range according to guide lines (US FDA,ANVISA,ICHQ.2B) ,then it is a validated method to demonstrate the Analyte concentrations in the sample.
Method Validation parameters:
The main purpose to perform the system suitability is to ensure that all analysing parameters of the method (reagents, samples, columns, instruments, glass ware, etc.) are suitable for the intended method. This experiment was performed by using injecting 6 subsequent injections of Aq MQC from a single vial.
Auto sampler carryover:
This parameter mainly used to ensure the carry over effect of initial injection to the subsequent injections during analysis. And can be performed by injecting the samples in the order of RS, AQ ULOQ, RS and AQ LLOQ of unextrated samples against STD BLK, AQ ULOQ, STD BLK and AQ LLOQ of extracted samples.
Linearity demonstrates the relationship between the experimental response values against analytical response values. Linearity graph can be plotted by using calibration curve standards. The number of standards used for constructing the calibration curve gives the information regarding the linearity range. The calibration consists of standard zero, eight or ten none zero standards. The calibration cure can plot by spiking the matrix with known concentration of the analyte. The calibration curve concentration range based on the expected concentration range of the particular study. The concentration of unknown sample can be identified by insert them in the calibration range.
The calibration curve of Analyte, Metabolites and ISTD was plotted by peak area ratio (Drug (r) Metabolites/ISTD) on Y-axis Vs the nominal concentrations on X-axis (first-order y = ax + b, where a=slope, b=intercept, x=concentration and y=peak area ratio of Analyte/ISTD).
LLOQ (Lower limit of Quantification): It is the lowest standard on the calibration curve.
ULOQ (Upper limit of Quantification): It is the higher standard on the calibration curve.
Precision is defined as the "Degree of the reproducibility or repeatability while doing the (experiment) measurements or calculations shows the similar the results.
Precision method is represented by % CV (coefficient of variation.
%CV= (SD/Mean) Ã-100
Accuracy is defined as the "degree of closeness of the experimental value to the true value.
% Accuracy = Obtained concentration of QC/Nominal concentration Ã- 100
% Mean Accuracy = mean of obtained Con.for QC/Nominal concentration Ã- 100
Both Accuracy and precision determined by two ways:
Within batch precision/ Accuracy
Between batch precision/ Accuracy
The % Accuracy for (STD2-STD10) should be within 85.00-115.00 %, for LLOQ should be within 80.00-120.00 %. ULOQ, LLOQ should pass and 75 % of CC standards (STD2-STD9) should meet the acceptance criteria. Response of interfering peaks in STD Blk at the retention time of ISTD should be ï‚£ 5.00 % of LLOQ.
For precision at least 67 % (16 out of 24) of total QC samples and 50 % (3 out of 6) at each level should pass .And the % CV â‰¤ 15% & for LLQC it should be â‰¤20%.
%Accuracy =obtained concentration of QC / Nominal concentration of QC Ã-100
%Mean Accuracy = mean of obtained concentration of QC / Nominal concentration
Of QC Ã-100
This parameter can be determined by performing specificity, matrix effect experiment.
It ensures that the intended method can able to differentiate the targeted analyte in the presences of other interfering substances.
It involves determination of any direct or indirect interference may alter the analytical response of the analyte. It can be performed by screening the different plasma lots. Reinjection Reproducibility:
This experiment can be carried out by injecting all ready passed P&A batch (1 set of CC and 6 sets of QC's) to identify again the P&A batches are giving same analytical results are not.
Effect of potential interfering drugs:
This experiment can preformed to ensure, any OTC drugs (e.g.: paracetamol, Ibuprofen, caffeine, diphinhydramine, diclofenacsodium and chlorfinaramine maleate.) may alter the Analytical response of the targeted Analyte.
This experiment can be preformed to ensure the extraction efficiency of the Analytical process and to know the recovery of the sample, by comparing the extracted sample area ratio against unextrated sample area ratio and reported as % recovery.
% recovery= Extracted analyte or ISTD peak ratio / unextrated analyte or ISTD peak area ratioÃ-100
Analyte peak area ratio= Area of analyte/Area of ISTD
ISTD peak area ratio=Area of ISTD/Area of Analyte
It defined as the degree of reproducibility of the method under a variety of normal method conditions.
Stability experiment procedure ensures the stability of analyte during sample collection, sample extraction; sample storage (i.e., Bench top, Freeze thaw, auto sampler, dry extract stability and solution stability).It can be performed by analysis of the Stability samples against comparative (freshly prepared) samples.