G1313a als

A reverse-phase HPLC method was developed in-house which allowed the simultaneous determination of the two active drugs in a single assay. This was achieved using an Agilent system comprised of a G1379B Degasser, G1311A QuatPump, G1313A ALS autosampler and G1314A VWD UV detector. An isocratic mobile phase of 80 % ammonium acetate (0.2 mol/L), 20 % acetonitrile plus 0.2 % w/v trifluoroacetic acid was used, along with a Kinetex 5 μm C18 150 x 4.6 mm column (Phenomenex, Macclesfield, UK). The detection wavelength was set at 254 nm, with injection volume 20 µL and flow rate 1.5 mL/min. Under these conditions the retention times of metronidazole and lidocaine were found to be approximately 1.5 and 3.0 min respectively. Calibration curves for metronidazole and lidocaine were constructed using concentrations in the range of 31.25 to 1000 µg/mL in methanol and both agents were fully baseline-resolved. An exemplar chromatogram is shown in Figure 2 and chromatographic parameters were determined as follows: k'metronidazole 6.5, k'lidocaine 14, α 2.15, Rs 4.6.

HPLC-DAD analyses were carried out using Agilent 1100 Series system (Agilent Technologies, Waldbronn, Germany) fitted with a thermostated autosampler (G1313A ALS) with a 500 μL loop, a quaternary pump (G1311A QUAT PUMP), degasser (G1322A) and with a diode-array detector (G1315A DAD). The evaluation of the collected data was made by ChemStation software (for LC 3D system Rev. B01.03 204, Agilent Technologies 2001-2005). Injection volume for each run was 200 mL. Flow rate was set to 0.6 mL/min, the column temperature was set to 25 °C and the acquisition wavelengths were 377 nm, 407 nm and 457 nm. Bilirubin and biliverdin were separated using a stainless steel BDS Hypersil C18 column (100 mm  4.6 mm I.D.) with a pre-column (4.0 mm  4.6 mm I.D.) with particle size of 2.4 mm and pore size of 120 Å (Thermo Fisher Scientific, Waltham, USA). The isocratic elution was made using 34% of 20 mM NH 4 OAc solution in MeOH.

This was used for the quantification of clonazolam in the tablets and to obtain product ions spectra for clonazolam and nitrazolam utilizing in-source CID.
Analyses were performed on an Agilent 1100 HPLC system equipped with a G13795 degasser, G1312A BinPump, a G1313A ALS and G1316A column oven (COLCOM) (Agilent, Little Island, Cork). Separation was obtained on an Allure PFP Propyl column (5 μm, 50 x 2.1 mm) Restek (Bellefonte, PA, USA). Mobile phase A consisted of 0.1% formic acid in water, whereas mobile phase B consisted of 0.1% formic acid in acetonitrile. The flow rate was set at 0.80 mL/min, with an injection volume of 10 µL, and the column temperature was 30°C. The following gradient elution program was used: 0-2 min 2% B, followed by an increase to 60% B within 15 min, followed by another increase to 80% B within 18 min before returning to 2% B within 25 min (total run time was 25 min.). The Agilent LC-MSD mass spectrometer settings were as follows: positive electrospray mode, capillary voltage 3500 V, drying gas (N 2 ) 12 L/min at 350 °C, nebulizer gas (N 2 ) pressure 50 psi, and scan mode m/z 70-500.
To obtain product ions spectra, samples were dissolved in acetonitrile/water (1:1, containing 0.1% formic acid) and the fragmentor voltage was set at 150 V. For the quantification of clonazolam in tablets, the fragmentor voltage was set at 50 V.