The keto acids contain a carbonyl group on adjacent carbon atom to carboxylic acid in PYR KB MKBA KG K3MVA K4MVA and PPY and reacted with DDB to form cyclic ring structure as substituted tetramethyl dihydro-pyrazinol (Fig.
3 4) (Table 2) with average recovery was 95 90 85 90 82 88 and 90 for PYR KB MKBA KG K3MVA K4MVA and PPY (n=4) respectively.
The results of analysis are Summarized in (Table 2) and were obtained within the concentration of g/ml PYR 6.12-7.36 KB 4.33-5.72 MKBA 3.45-6.32 KG 3.83-6.49 K3MVA .53-7.78 K4MVA 4.46-7.38 and PPY 9.08-9.71 with RSD1.1-1.9% 1.2-1.8% 1.4-1.9% 1.1- 2.4% 1.3-3.0% and 1.3-2.8% respectively.
The results obtained (Table 3) indicatedg/ml PYR 0.11-0.24 KB 0.15-0.26 MKBA 0.11-0.38 KG 0.12-0.23 K3MVA 0.12-0.34 K4MVA 0.22-0.39 and PPY 0.82-1.3 with RSD 1.0-2.0% 0.6-1.3% 1.0-1.9% 1.2-2.1% 0.9-1.5% 1.1-2.6% 1.2-3.1% respectively.
HPLC analysis of PYR KBA MKBA KG K3MVA K4MVA and PPYR from serum of healthy volunteers.
A solution was processed as HPLC analytical procedure and others were added MKBA 50 ug, K3MVA 60 ug, and K4MVA 100 ug, PPY 40 ug, and again processed as HPLC analytical procedure.
A part was treated as HPLC analytical procedure and other was added PYR (3 ug), KB (8 ug), MKBA (6 ug), K3MVA (30 ug), KG (30 ug), K4MVA (60 ug), PPY (40 ug), and procedure HPLC was again followed the final volume was adjusted to 5 mL.
A sample was processed as HPLC procedure and other was added PYR (2 ug), KB (6 ug), MKBA (8 ug), K3MVA (40 ug), KG (30 ug), K4MVA (70 ug) and PPY (60 ug), again processed as HPLC procedure.
Nine a-keto acids: GA, PYR, KB, MKBA, K3MVA, KG, K4MVA, KHA, and PPY were treated with NPD separately and the derivatives formed were extracted in chloroform, and examined for elution from HPLC column.
An isocratic elution with methanol: water: acetonitirle (42: 56: 2, v/ v/ v) with a flow rate of 0.9 mL/ min resulted a better resolution of GA PYR, KB, MKBA, K3MVA, KG,K4MVA, KHA, and PPY peaks.
Linear calibration curves were obtained by recording average peak height/ peak area (n = 4) versus concentration of GA (0.2-5.0 ug/ mL), PYR (0.2-20 ug /mL), KB (0.3-100 ug/ mL), MKBA (0.45-100 ug/mL), KG (0.5-100 ug/ mL), K3MVA (0.6-100 ug/ mL), K4MVA (0.6-100 ug/ mL), KHA (0.7-100 ug/ mL), PPY (0.7-100 ug/ mL) with coefficient of determination (r2) ranging 0.9953-0.9988.
The amounts of MKBA, K3MVA, K4MVA , and PPY found per tablet were 82.5 mg, 64.8 mg, 98 mg, and 65.8 mg, with a RSD of 2.1%, 2.6%, 2.2% and 1.9%, respectively.