Determination of Vitamin B12 in food products and in premixes by reversed-phase high performance liquid chromatography and immunoaffinity extraction.

A new, faster and simple method to quantify Vitamin B12, both in foods and in premixes, by reversed-phase liquid chromatography with UV detection has been developed. Vitamin B12 was extracted from food products with 50 mM sodium acetate buffer pH 4.0 (at 100 degrees C for 35 min) in the presence of sodium cyanide, followed by a purification step on an immunoaffinity column prior to the LC analysis. An enzymatic hydrolysis (pepsin at 37 degrees C and pH 4 for 3 h) prior to the purification step efficiently released the bound Vitamin B12, and thus, allowed obtaining total Vitamin B12 content in food products. Vitamin B12 was monitored by UV at 361 nm after its separation on a reversed-phase narrow-bore column with a gradient of mobile phase made of water/acetonitrile and trifluoroacetic acid (TFA) 0.025%. The specificity of the method was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity with the Vitamin B12 standard. The calibration graphs plotted with six concentrations of Vitamin B12 was linear with a regression coefficient R2 > 0.9997. The repeatability of the method was evaluated at different levels of concentration on six fortified products and the relative standard deviation (RSDr) was below 3.2%. The value of the relative standard deviation of the intermediate precision was below 5.6% (n = 4). The method was successfully applied to several food products and consistent results were obtained in comparison with microbiological assay (MBA). Our data demonstrate that the immunoaffinity columns are highly efficient for the purification of Vitamin B12 and that our HPLC could be used as an alternative method to the microbiological assay for the determination of Vitamin B12 in food products.

Chemical instability and methods for measurement of cisplatin adducts formed by interactions with cysteine and glutathione.

Reactions between cisplatin or its aquated species and L-cysteine (L-cys) or glutathione (GSH) were studied in vitro using liquid chromatography on-line with inductively coupled plasma mass spectrometry (LC-ICPMS) and/or electrospray ionization mass spectrometry (LC-MS) in order to obtain information on the mechanisms occurring in treated patients. Reaction between cisplatin and L-cys yielded initially 4 adducts of which only 2 were stable and detectable after 24 hours incubation; their structures corresponded to bis-platinum cysteinyl adducts. Reaction of cisplatin with GSH proceeded via the formation of at least 11 glutathione-platinum adducts (G1 - G11) which underwent parallel reactions within 24 hours of incubation, probably to form higher molecular weight species. Of the 11 adducts, only 2, G3 and G7, whose structures correspond to [Pt(NH3)2Cl]2(SG) and [Pt(NH3)2OH]2(SG) were still present in the reaction mixture after 24 hours incubation. This study shows that GSH, and to a lesser extent L-cys, incubated with cisplatin in vitro forms unstable and reactive platinum compounds and that LC-ICPMS and LC-MS are 2 complementary techniques suitable for the study of organometallic compounds.

Comparison of the reactivity of oxaliplatin, pt(diaminocyclohexane)Cl2 and pt(diaminocyclohexane1)(OH2)2(2+) with guanosine and L-methionine.

The initial rates of reactivity of oxaliplatin, its metabolites Pt(dach)Cl2 and Pt(dach)(OH2)2(2+) with guanosine and L-met in water, NaCl and phosphate were compared. Versus guanosine, the most reactive molecule was Pt(dach)(OH2)2(2+), about 40 fold that of oxaliplatin, the least reactive was Pt(dach)Cl2, Versus L-met, Pt(dach)(OH2)2(2+), was also the most reactive species but only about 2 fold more reactive than Pt(dach)Cl2 and oxaliplatin. Pt(dach)(OH2)2(2+) was approximately 3 fold less reactive versus methionine than guanosine whereas oxaliplatin and Pt(dach)Cl2 were about seven fold more reactive versus methionine than guanosine. Thus, the three platinum compounds oxaliplatin, Pt(dach)Cl2 and Pt(dach)(OH2)2(2+) react with L-met but only the Pt(dach)(OH2)2(2+) has a high reactivity with guanosine. Oxaliplatin, which is stable in water, has to be transformed in the presence of chloride in chloro-derivatives which are aquated to become active particularly versus guanosine. These data demonstrate that oxaliplatin has similarities with cisplatin in terms of chloride versus water coordination and in terms of dependence on chloride concentration for transformations.

Early biotransformations of oxaliplatin after its intravenous administration to cancer patients.

This article deals with the fate of oxaliplatin 1 and 3 h after its i.v. administration (130 mg/m(2)) to three patients. Its binding to plasma proteins and penetration into red blood cells were monitored by chromatography on-line with inductively coupled plasma mass spectrometry. Oxaliplatin biotransformations in plasma ultrafiltrate (PUF) and in urine were studied by chromatography coupled to inductively coupled plasma mass spectrometry or to electrospray ionization mass spectrometry. In plasma, four platinum (Pt) compounds were found. The peaks at 200 and 160 kDa corresponding to gamma-globulins contained 40% of the Pt bound; the peak at 60 kDa corresponding to albumin contained 40% of the Pt found. The peak <2 kDa could correspond to oxaliplatin, to its degradation products, or to adducts between Pt compounds and low-molecular-weight species such as glutathione, L-methionine, and L-cysteine. In PUF and urine, oxaliplatin itself, its degradation products, Pt(dach)Cl(2), [Pt(dach)(OH(2))Cl](+), and species that have the same retention times as Pt(dach)(methionine) and [Pt(dach)](2)(glutathione) were found. One hour after infusion, oxaliplatin in PUF and urine represented 12 and 50% of the total Pt, respectively. Three hours after infusion, oxaliplatin, undetectable in PUF, represented 10% of total Pt in urine. Inside red blood cells, two Pt compounds were found. The Pt peak at 60 kDa corresponding to hemoglobin and the peak <2 kDa corresponding to low-molecular species contained, respectively, 60% and 40% of Pt found. This study demonstrates that in the first hours after its infusion, oxaliplatin, in addition to other Pt compounds, is present in plasma and urine and that Pt is bound to albumin, gamma-globulins, and hemoglobin.

Mechanisms of reaction of L-methionine with carboplatin and oxaliplatin in different media: a comparison with cisplatin.

The activity of platinum compounds is dependent on nucleophile substitution reactions. In this paper, we study the reactivity of L-met with carboplatin, oxaliplatin and cisplatin by following with HPLC-UV the concentration of L-met and by characterizing the resulting adducts with LC-MS. In the absence of NaCl, in water, the initial rate at which L-met concentration decreases with cisplatin, oxaliplatin and carboplatin is 0.25 +/- 0.007, 0.057 +/- 0.01 and 0.17 +/- 0.02 mM h(-1), respectively. In phosphate buffer this rate is 0.056 +/- 0.009 for cisplatin, 0.019 +/- 0.001 and 0.13 +/- 0.02 for carboplatin and oxaliplatin, respectively. Reactions of L-met with cisplatin occurred via its conversion into monoaqua species in water and into phosphato-derivatives (AP) in phosphate buffer but finally the same methionine-platinum adducts M2 [(NH3)2(met)]Pt, M4 and M5 [(met)2]Pt were characterized. Reaction of carboplatin with L-met occurred via the formation of M0 [(NH3)2(met)(CBDCA)]Pt whose structure is consistent with the direct interaction of L-met with carboplatin. However, the same final products as those found with cisplatin were characterized. The reaction of oxaliplatin with L-met proceeded through a mechanism similar to that of carboplatin to give M7 [(met)(DACH)]Pt. In the presence of NaCl, cisplatin directly reacted with L-met to yield at least five methionine-platinum adducts. The reaction of carboplatin gave the same adducts suggesting its transformation into cisplatin. The reaction of oxaliplatin with L-met occurred via the formation of aquated species A [(OH)(Cl)(DACH)]Pt which readily underwent reaction with L-met to form M6 [(met)(Cl)(DACH)]Pt and M7. This study shows that the reactivity of cisplatin, carboplatin and oxaliplatin is dependent on the media in which they occur. The discrepancy between their reactions with L-met could partly explain their therapeutic differences.