Development and Validation of an LC/MS Method for Quantitative Determination of Thiamine in Blood Plasma
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STRUCTURE OF CHEMICAL COMPOUNDS, METHODS OF ANALYSIS AND PROCESS CONTROL DEVELOPMENT AND VALIDATION OF AN LC/MS METHOD FOR QUANTITATIVE DETERMINATION OF THIAMINE IN BLOOD PLASMA O. A. Tashirova,1,2 G. V. Ramenskaya,1 A. M. Vlasov,1 and M. R. Khaitov2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 46, No. 12, pp. 46 – 48, December, 2012. Original article submitted September 14, 2012.
A new sensitive method based on liquid chromatography with mass-spectrometric detection (LC/MS) was developed and validated for the quantitative determination of thiamine in blood plasma. The method is highly sensitive and selective due to the combination of LC and MS and can be used for determining the thiamine blood concentration during pharmacokinetic investigations. Keywords: thiamine, blood plasma, liquid chromatography/mass spectrometry
Because all forms of thiamine are converted biochemically into thiamine species with various degrees of phosphorylation that exist in the blood in chemical equilibrium with free thiamine, we decided to develop a rapid and simple method for quantitative determination of thiamine in blood plasma in order to study the comparative pharmacokinetics of water-soluble and lipid-soluble forms of thiamine.
Vitamin B1 is a water-soluble vitamin. However, both water-soluble and lipid-soluble forms are used in pharmacotherapy. The water-soluble forms include thiamine salts such as thiamine hydrochloride, bromide, and mononitrate [1]. Preparations containing water-soluble thiamine salts are currently used less often in therapy because these preparations typically have certain drawbacks including low bioavailability that is less than 10% of the administered dose, poor permeability through intestinal mucous membranes, rapid metabolism in the gastro-intestinal (GI) tract, and a high degree of metabolism in the liver and tissues [2 – 4]. Lipid-soluble forms of thiamine were synthesized during the 1960s in order to solve this problem. These included benfothiamine (S-benzoylthiamine-O-monophosphate); fursulthiamine (thiamine tetrahydrofurfuryl disulfide); octothiamine; S-acylthiamine; O,S-diacetylthiamine; cyclothiamine; prosulthiamine; sulbuthiamine; and others (Fig. 1) [4 – 8]. Lipid-soluble forms of thiamine penetrate rather quickly and completely into intestinal epithelial cells upon absorption from the GI tract and are converted there into ordinary thiamine [9]. 1 2
EXPERIMENTAL PART We used thiamine hydrochloride (99.92% pure, Supelco, Germany, Cat. No. 47858), trifluoroacetic acid (TFA) and CH3CN (Merck KGaA, Darmstadt, Germany), de-ionized and purified H2O using an MP-650 Milli-Q system (Iwaki Millipore, Japan). Thiamine in blood plasma was determined on an Agilent 1200 LC/MS equipped with a degasser, gradient pump, column thermostat, autosampler, UV detector, and Agilent 6120 quadrupole mass-spectrometric detector with electrospray ionization at atmospheric pressure. The stationary phase was an Agilent Zorbax SB C18 chromatography column (5 mm, 150 ´ 2.1 mm) with a Zorbax SB C18 precolumn (5 mm,
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