Hplc grade acetonitrile

Saliva samples were cold centrifuged at 402 ×g (3000 rpm) and decanted into fresh tubes for snap freezing in liquid nitrogen and stored at −20°C. Arecoline level quantification was done using high-performance LC-MS/MS method, using following materials: AB SCIEX API 4000™ LC-MS/MS System, Foster City, CA/Concord, Ontario, Canada, Arecoline hydrochloride, Arecoline hydrobromide and Triethylamine from Sigma-Aldrich (Germany), HPLC grade – Acetonitrile, Methanol and Water and Research grade sodium hydrogen phosphate from Spectrochem Pvt., Ltd. (India). A procedure based on LC-MS/MS was used for arecoline quantification. Liquid/liquid extraction with chloroform/isopropanol (95:5 v/v) was used for extraction procedure.
Chromatography was performed on a C8 reversed-phase column using a gradient of 50-mM ammonium formate, pH 5.0 and acetonitrile as a mobile phase at a flow rate of 0.5 mL/min. Separated analytes were determined by electrospray ionization MS/MS in the positive ion mode using multiple reaction monitoring.

The samples of isoproterenol hydrochloride obtained from different final step batches, reference standard of isoproterenol hydrochloride (purity 99.9%) and the starting material, 1-(3,4-bis(benzyloxy)phenyl)-2-(isopropylamino)ethanone hydrochloride (2) used in the synthesis, were provided by Chemical Research Division, Micro Labs Ltd. (Bangalore, India). The catalyst, Pd-C (50% wet), was acquired from Monarch Catalyst Private Limited (Mumbai, India). HPLC grade acetonitrile and methanol were purchased from Spectrochem Pvt Ltd. (Bangalore, India), and formic acid and trifluoroacetic acid were purchased from Sigma-Aldrich (Bangalore, India). The commercial grade ethyl acetate (EtOAc) and isopropyl alcohol (IPA) were procured from Taiwan Fieldrich Corporation (Taipei, China). Purified water by Millipore Milli-Q Plus Purification System (Bradford, PA, USA) was used during the course of experimental studies. CD₃OD was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). Potassium bromide used in Fourier transform infrared spectroscopy (FT-IR) was purchased from Merck KGaA (Darmstadt, Germany). TLC plates (60 F₂₅₄) procured from Merck (Delhi, India) was used to check the progress of reaction.