Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/89659
Type: Artigo de periódico
Title: Hydroxocobalamin Quantification In Human Plasma By High-performance Liquid Chromatography Coupled With Electrospray Tandem Mass Spectrometry In A Pharmacokinetic Study
Author: Mendes G.D.
Mendes F.D.
Sampaio M.F.
Silveira A.S.
Chen L.S.
Alkharfy K.M.
De Nucci G.
Abstract: 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.
Editor: 
Rights: aberto
Identifier DOI: 10.4172/jbb.1000139
Address: http://www.scopus.com/inward/record.url?eid=2-s2.0-84876076370&partnerID=40&md5=db94144cbd472fe8718db29db35e7db4
Date Issue: 2013
Appears in Collections:Unicamp - Artigos e Outros Documentos

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