A rapid, sensitive and specific method for quantifying hydroxocobalamin in human plasma using paracetamol as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethanol 100%; -20°C). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on Prevail C8 3 μm, analytical column (2.1×100 mm i.d.). The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 5-400 ng.mL -1 (r>0.9983). The limit of quantification was 5 ng.mL-1. The method was also validated without the use of the internal standard. The precision in the intra-batch validation with IS was 9.6%, 8.9%, 1.0% and 2.8% whereas without IS was 9.2%, 8.2%, 1.8% and 1.5% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in intra-batch validation with IS was 108.9%, 99.9%, 98.9% and 99.0% whereas without IS was 101.1%, 99.3%, 97.5% and 92.5% for 5, 15, 80 and 320 ng/mL, respectively. The precision in the inter-batch validation with IS was 9.4%, 6.9%, 4.6% and 5.5% whereas without IS was 10.9%, 6.4%, 5.0% and 6.2% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in inter-batch validation with IS was 101.9%, 104.1%, 103.2% and 99.7% whereas without IS was 94.4%, 101.2%, 101.6% and 96.0% for 5, 15, 80 and 320 ng/ mL, respectively. This HPLC-MS-MS procedure was used to assess the pharmacokinetics of cobalamin following intramuscular injection 5000 μg in healthy volunteers of both sexes (10 males and 10 females). The volunteers had the following clinical characteristics (according to gender and expressed as mean ± SD [range]): males: age: 32.40 ± 8.00 [23.00-46.00], height: 1.73 ± 0.07 m [1.62-1.85], body weight: 72.48 ± 10.22 [60.20-88.00]; females: age: 28.60 ± 9.54 [18.00-44.00], height: 1.60 ± 0.05 [1.54-1.70], body weight: 58.64 ± 6.09 [51.70-66.70]. The following pharmacokinetic parameters were obtained from the hydroxocobalamin plasma concentration vs. time curves: AUC last, T1/2, Tmax, Vd, Cl, C max and Clast. The pharmacokinetic parameters were 120 (± 25) ng.mL -1 for C max, 2044 (± 641) ng.hr.mL -1 for AUClast, 8 (± 3.2) ng.mL -1 for Clast, 38 (± 15.8) hr for T 1/2 and 2.5 (range 1-6) hr for Tmax. Female volunteers presented significant (p=0.0136) lower AUC (1706 ± 704) ng.hr.mL-1) and larger (p=0.0205) clearance (2.91 ± 1.41 L/hr), as compared to male 2383 ± 343 ng.hr.mL -1 and 1.76 ± 0.23 L/hr, respectively. These pharmacokinetic differences could explain the higher prevalence of vitamin B12 deficiency in female patients. The method described validated well without the use of the internal standard and this approach should be investigated in other HPLC-MS-MS methods. © 2013 Mendes GD, et al.528087Butler, C.C., Vidal-Alaball, J., Cannings-John, R., McCaddon, A., Hood, K., Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency: A systematic review of randomized controlled trials (2006) Fam Pract, 23, pp. 279-285Kuzminski, A.M., Del Giacco, E.J., Allen, R.H., Stabler, S.P., Lindenbaum, J., (1998) Blood, 92, p. 1191van Asselt, D.Z., Merkus, F.W., Russel, F.G., Hoefnagels, W.H., Nasal absorption of hydroxocobalamin in healthy elderly adults (1998) Br J Clin Pharmacol, 45, pp. 83-86Van den Berg, M.P., Merkus, P., Romeijn, S.G., Verhoef, J.C., Merkus, F.W., Hydroxocobalamin uptake into the cerebrospinal fluid after nasal and intravenous delivery in rats and humans (2003) J Drug Target, 11, pp. 325-331Merkus, P., Guchelaar, H.J., Bosch, D.A., Merkus, F.W., (2003) Neurology, 6, p. 1669Chen, J.H., Jiang, S.J., Determination of cobalamin in nutritive supplements and chlorella foods by capillary electrophoresis-inductively coupled plasma mass spectrometry (2008) J Agric Food Chem, 56, pp. 1210-1215Kelly, R.J., Gruner, T.M., Sykes, A.R., Development of a method for the separation of corrinoids in ovine tissues by HPLC (2005) Biomed Chromatogr, 19, pp. 329-333Rosin, H., Man, W.Y., Doyle, E., Bult, A., Beijnen, J.H., (2000) J Liq Chromatogr and Related Tech, 23, p. 329Wang, Y.H., Yan, F., Zhang, W.B., Ye, G., Zheng, Y.Y., (2009) Neurosci Bull, 25, p. 209Dubois, D., Dubois, E.F., (1916) Arch Intern Med, 17, p. 862Mendes, F.D., Chen, L.S., Borges, A., Babadópulos, T., Ilha, J.O., Ciprofibrate quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry for pharmacokinetic studies (2011) J Chromatogr B Analyt Technol Biomed Life Sci, 879, pp. 2361-2368Uhl, W., Nolting, A., Gallemann, D., Hecht, S., Kovar, A., Changes in blood pressure after administration of hydroxocobalamin: Relationship to changes in plasma cobalamins-(III) concentrations in healthy volunteers (2008) Clin Toxicol, 46, pp. 551-559. , (Phila)