It is a metabolic product of bromhexine. Ultraviolet-visible spectrophotometric method has been reported for the quantitative determination of CEFPO from pharmaceutical formulation spectrophotometric method��[1] using high performance liquid chromatography (HPLC),[2�C4] and high performance thin layer chromatography (HPTLC).[5] A method for the simultaneous determination exactly of AMBRO has not been reported with CEFPO such as UV spectrophotometry,[6] HPTLC[7] and HPLC[8] and in human plasma using LC-MS/MS.[9] No simultaneous estimation method was developed for determination of CEFPO and AMBRO in human plasma. Therefore, a simple, sensitive, rapid, and economic HPTLC method has been developed for the determination of CEFPO and AMBRO in human plasma using paracetamol as an internal standard.
Figure 1 (a) Structure of cefpodoxime proxetil, (b) Paracetamol, (c) (IS), and ambroxol hydrochloride MATERIALS AND METHODS Instrumentation HPTLC Camag with precoated silica gel Plate 60F254 (20 cm ��10 cm) 250 ��m thicknesses (E. Merck, Darmstadt, Germany) was used as the stationary phase. Sample application was done by using a Camag 100 ��l syringe and a Camag Linomat V applicator. The sample was sprayed in the form of narrow bands of 8 mm length at a constant rate 2 ��l/s. Linear ascending development was carried out in a 20 cm �� 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland). The densitometric scanning was performed by using a Camag TLC scanner III supported by win CATS software (V1.4.2.8121 Camag). Chromatogram was evaluated by using a ratio of peak areas of drugs with an internal standard.
Chemicals CEFPO (Blue Cross Laboratories Ltd., Ambad Nashik, India), AMBRO (Blue Cross Laboratories Ltd., Ambad Nashik, India), and paracetamol (Kirti Pharmachem Ltd., Sinner, Maharashtra, India) were received having 98.80%, 98.70% and 100.1% purity, respectively. They were used as such without checking their purity. The HPLC grade methanol and Analytical reagent grade chloroform were purchased from S D Fine Chem. Ltd., Mumbai, India. Human plasma used for research work was supplied by Arpan Blood Bank, Nashik, Maharashtra, India. Preparation of stock solution and working standard solution Stock solutions 1.0 mg/ml each of CEFPO, AMBRO and paracetamol were prepared in methanol.
Preparation of plasma sample In a 15 ml centrifuge tube 10, 20, 30, 40, 50, and 60 ��l of working stock solution of CEFPO were added to drug-free plasma to provide calibration standards of 500, 1000, 1500, 2000, 2500, and 3000 ng/ml and 2, Carfilzomib 4, 6, 8, 10, and 12 ��l of working stock solution of AMBRO were added to drug-free plasma to provide calibration standards of 1000, 2000, 3000, 4000, 5000, 6000 ng/ml and 1000 ng/ml of paracetamol (internal standard) was kept constant. The quality control (QC) samples were prepared in plasma in the concentration range 1000, 2000, 3000 ng and 2000, 4000, 6000 ng for CEFPO and AMBRO.