Dionex Ultimate 3000 liquid chromatograph (Dionex, USA); Linseis DSC-PT10 differential scanning calorimetry instrument (Germany); Sartorius pB-10 pH meter; Brucker AVANCEⅡ400 NMR spectrometer (Bruker, USA); Mettler Toledo AB135-S electronic balance (Mettler, German); Shimadzu AUY120 electronic balance; Millipore pure water instrument.

Octane sulfonate (batch no. 30204861), mannitol (batch no. 980708) and phosphoric acid (batch no. 10015418) were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The maleic acid (purity 99.0%) was purchased from Sigma, USA. D2O (D, 99.9%) was from Cambridge Isotope Laboratories Inc., UK. Acetonitrile was HPLC grade from Merck, German. High-purity water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA, Milli-Q filtered, 18.2 MΩ/cm). The meclofenoxate hydrochloride bulk drug was from Shanghai Wandai Pharmacaceutical Co., Ltd. The meclofenoxate hydrochloride in sterile powder for injection was from Hunan Wuzhoutong Pharmaceutical Co., Ltd. (Hunan, China).

The experiment was performed using an online degasser, an autosampler and a Phenomenex luna C18 Column (5 μm, 250 mm × 4.6 mm i.d.). The chromatographic elution was accomplished isocratically with Octanesulfonate solution (0.05 mol/l, pH 2.5)-ACN (65:35 v/v) at a flow rate of 1 ml/min. The temperature was maintained at 30˚C and the injection volume was 20 μL. DAD detection was achieved in the range of 200 - 800 nm, 225 nm results were used for quantitative purposes.

Octanesulfonate solution (0.05 mol/l, pH 2.5): Weigh 10.81 g of Octanesulfonate, transfer into a beaker, add an appropriate amount of water to dissolve, adjust to pH 2.5 with phosphoric acid solution and add water to make 1000 ml. The prepared solution was filtered with 0.22 μm membrane.

Mixed solvent (ACN:H₂O = 6:4): Mix 200 ml of water adjusted to pH 2.5 with phosphoric acid solution with 300 ml of acetonitrile.

Weigh 25 mg of meclofenoxate hydrochloride bulk drug, transfer into a 25 ml volumetric flask, add acetonitrile to a constant volume is 25 ml and filter with 0.45 μm organic membrane to make 1 mg/ml meclofenoxate hydrochloride solution. Take appropriate amounts of the solution in 10 ml volumetric flasks to make 0.5, 0.25, 0.125, 0.0625, 0.0312 mg/ml meclofenoxate hydrochloride standard solution.

Weigh 25 mg of meclofenoxate hydrochloride in sterile powder for injection, transfer into a 25 ml volumetric flask, add mixed solvent to a constant volume is 25 ml and filter with 0.45 μm organic membrane.

Nitrogen (99.999% purity) was the purge gas flowing at 10 ml/min. Samples of 1 - 3 mg were weighed into aluminum pans and covers were hermetically sealed into place. An empty, hermetically sealed aluminum pan was used as reference. Samples were preheated at 50˚C and were heated to 250˚C at a rate of 10˚C/min.

Sample solutions were 29.60 mg/ml and the internal standard solution of maleic acid was 2.63 mg/ml; the solvent was D₂O. The experiments were carried out with the following parameters optimized for qNMR: 90˚ pulse of 11.10 usec, delay of 20 s, 16 scans and gain of 10. Phase and baseline corrections were done manually.

Six concentrations ranging from 0.0312 mg/ml to 1.0 mg/ml were used to obtain the calibration curve. A straight line with an excellent correlation coefficient with negligible deviations from linearity at low and high concentrations was obtained. The linear equation was Y = 712.13X − 1.5178 (R² = 1). X is concentration and Y is peak area. The UV chromatogram of meclofenoxate hydrochloride in sterile powder for injection is shown in Figure 2. The content of meclofenoxate hydrochloride in sterile powder for injection is 38.8% calculated by the linear equation.

The meclofenoxate hydrochloride bulk drug was determined in the different weight to get the relationship between the melting energy and the weitht of meclofenoxate hydrochloride. Figure 3 gave the calibration curves as the normalized area of the melting peaks (Energy) and plotted against the weight of meclofenoxate hydrochloride. The energy of meclofenoxate hydrochloride is proportional to its weight (range from 1.0 to 3 mg). The linear equation was y = 136.31x + 5.6819. The y is energy and the x is weigh. The correlation coefficient of the best-fit line was 0.9982, indicating a linear correlation between the energy and the weight of meclofenoxate hydrochloride. Based on this single set of data, DSC appeared to be a suitable technique for the detection of meclofenoxate hydrochloride.

The mixtures of meclofenoxate hydrochloride and mannitol were made over the range of 35% to 52% w/w meclofenoxate hydrochloride respectively. The content of meclofenoxate hydrochloride was determined by DSC method. Table 1 gave a comparison between the theoretical meclofenoxate hydrochloride content and determined meclofenoxate hydrochloride content in mixtures by DSC method. The recoveries of meclofenox-ate hydrochloride in sterile powder for injection were from 98.3% to 102.3% (n = 3).

Figure 4 gave DSC curves of the meclofenoxate hydrochloride in sterile powder for injection. The content of meclofenoxate hydrochloride in sterile powder for injection was 39.3% by the linear equation of quantitative DSC method.

For quantitative methods, the internal standard method is used widely at present. The purity of the analyte can be calculated as follows [9] :

where M_x and M_Std are the molar masses of the analyte and the standard, respectively, m is the weighed mass of the investigated sample, m_Std and P_Std are the weighed mass and the purity of the standard, and N_Std and I_Std correspond to the number of spins and the integrated signal area of a (typical) NMR line of the standard.

The most important conditions for an internal standard are its solubility and its chemical interaction with the analyte. Maleic acid is easily solved in D₂O and the chemical shift is 6.319 which best separated with meclofenoxate showed in Figure 5. Furthermore, there is no chemical interaction between them. Therefore, maleic acid is selected as the internal standard.

The ¹H NMR data of meclofenoxate hydrochloride in sterile powder for injection is shown in Table 2. The monitor signal is chosen as the one that is best separated from the other signals for meclofenoxate in the experiment. Five monitor signals of meclofenoxate are selected for quantization (Table 3). As shown in Table 3, the content of meclofenoxate hydrochloride in sterile powder for injection was 39.1% by the internal standard method of quantitative NMR. The quantitation of meclofenaxate was reproducible and the relative standard deviation is 1.0%.

We compare the different method for the determination of meclofenoxate hydrochloride for injection in Table 4. According to the result of HPLC, the content of meclofenoxate hydrochloride determinated by the other two methods (DSC and NMR) was accurate.