Abstract:

A method was established for the determination of N -nitrosodimethylamine (NDMA) in metformin hydrochloride active pharmaceutical ingredient (API) and preparation samples by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Water was used as the extraction solvent for the metformin hydrochloride API and preparation samples. The samples were analyzed by HPLC-MS/MS after vortex mixing, constant temperature shaking, high speed centrifugation and microfiltration. An ACE EXCEL 3 C18-AR column (150 mm×4.6 mm, 3 μm) was used for chromatographic separation. The mobile phases were water and methanol both containing 0.1% formic acid with gradient elution. The flow rate, column temperature, and autosampler temperature were set as 0.8 mL/min, 40℃, and 10℃, respectively. The valve switching technique was used to protect the mass spectrometer, while six-way valve switching was adopted to allow the mobile phase with a retention time of 2.85-7.00 min to enter the mass spectrometer and the mobile phase with other retention times to enter the waste liquid. For the mass spectrometer, an atmospheric pressure chemical ionization (APCI) ion source was used in positive ion MRM scanning mode. The other conditions were as follows:atomizer flow, 3 L/min; heater flow, 10 L/min, interface temperature, 300℃; desolvation line (DL) temperature, 250℃; heating block temperature, 400℃; and dryer flow, 10 L/min. The quantitative transition of NDMA was m /z 75.0→43.1 with a collision energy of-17.0 eV, while the qualitative transition was m /z 75.0→58.2 with a collision energy of-16.0 eV. The external standard method was utilized for quantitative analysis. The established method was validated in detail by investigating the specificity, linear range, limit of detection, limit of quantification, recovery, precision, and stability. This method showed good specificity, since the solvents and excipients did not interfere with the determination of NDMA. A good linear relationship was observed the NDMA peak area and the mass concentrations in the range of 1.00-100.00 ng/mL with an excellent correlation coefficient (r >0.9999). The limit of detection and limit of quantification in solution were 0.20 ng/mL and 1.00 ng/mL, respectively. The recoveries of NDMA at low, medium, and high spiked levels ranged from 94.55% to 114.67%, and the RSDs ranged from 4.73% to 13.46%, indicating good accuracy and precision for the quantification of NDMA. Stability tests showed that NDMA was stable when placed in the autosampler for 0, 8, 24 h, since the RSD of the peak area was as low as 2.08%. The validated method was then applied to the determination of NDMA in metformin hydrochloride raw materials and preparations (tablets, capsules or enteric tablets). The detected amount of NDMA in the API did not exceed the limit in 113 batches of samples, but NDMA was detected and exceeded the limit in eight batches of preparations. This method is sensitive, accurate, and easy to operate, and it can be used for the determination of NDMA in metformin hydrochloride raw materials and preparation samples.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), trace determination, N -nitrosodimethylamine (NDMA), metformin