3.1. Standard Calibration (CC) and Quality control

Reagents and Solvents Preparation

All the solvents used in this research were
HPLC grade and regents were AR-grade. The volumes and concentrations were
mentioned in the process are theoretical. The volumes and concentrations could
be corrected on the basis of actual weights used.

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Reagents and solvents preparation of DG and SG


Table-2:  Stock solution preparation of DG, SG, DGd5
and SGd2

Name of the stock solution


Volume (mL)



3.2. Standard Calibration (CC) and Quality control
(QC) Samples Preparation

Standard Stock solutions of DG (100.0 µg.mL-1),
DGd5 (100.0 µg.mL-1), SG (100.0 µg.mL-1), SGd2 (100.0
µg.mL-1) were prepared in methanol. From each stock solution 100.0
ng.mL-1 intermediate dilution was prepared in plasma. Aliquots of
100.0 ng.mL-1 were used to spike blank human plasma in order to
obtain calibration curve standards of 50.0, 100.0, 500.0, 1000.0, 2000.0,
4000.0, 6000.0, 8000.0, 10000.0 pg/mL. Four levels of QC concentrations at
50.0, 150.0, 3000.0 and 7000.0 pg/mL (LLOQ, LQC, MQC and HQC) were prepared by
using the different plasma. Spiked calibration curve standards and Quality
control standards were stored at -30oC. Standard stock solutions of DGd5,
SGd2 (100.0 µg.mL-1) were prepared in methanol . DGd5 and
SGd2 was further diluted to 10.0 ng.mL-1 (Spiked
concentration of internal standard) using 50 % methanol and stored in the
refrigerator 2-8 0C until analysis.


During bioanalytical
method development HPLC,
mass spectrometric and sample extraction parameters were optimized to achieve
the best results.

4.1. Development of HPLC Parameters

During the development stage HPLC conditions
like column, mobile phase and internal standard were optimized in a logical and
sequential manner.

4.2. Development of Mass

The MS optimization was performed by direct infusion of solutions
of SG, DG, DGd5 and SGd2 into the ESI source of the mass spectrometer. The
vital parameters like ionization type, temperature, voltage, gas parameters
such as nebulizer and heater gases, compound parameters like DP, EP, FP, CE and
CXP were optimized to obtain a better spray shape and ionization to form the
respective productions from the protonated SG, DG, DGd5 and SGd2 molecules .The mass
transitions were selected as m/z 410.2/250.6,
415.3/250.6, 316.1/272.4 and m/z 318.2/272.3 for quantification of DG,
DGd5, SG and SGd2 respectively.

4.3. Development of Extraction Solvent and Buffer

Various organic solvents and buffers were
optimized to extract DG and SG from plasma
samples. After a series of trials, ethyl acetate and 5mM NaH2PO4
buffer were selected as appropriate due to high recovery efficiency and
matrix free interference.

Sample Extraction and Cleanup Procedure (Sample

Liquid-liquid extraction was carried out to extract the
drug and IS for this purpose 100 µL of respective concentration of plasma
sample was taken into polypropylene tubes 
and mixed with 50µL of internal standard (10.0 ng.mL-1). This
was followed by addition of 100 mL of 5mM NaH2PO4
solution and 3.0 mL of ethyl acetate
and vortexed for approximately 10 minutes. Then the Samples were centrifuged at
4000 rpm for 10 minutes at 20°C.
Further, the supernatant was
transferred into labeled polypropylene
tubes and evaporated with nitrogen gas at 40°C.  Then the samples were
reconstituted with the mobile phase and vortexed for 2 minutes.  Finally, Sample was transferred into auto
sampler vials to inject into the LC-MS/MS.


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