Franco M. Pasutto

Gas chromatographic analysis of trace phenols by direct acetylation in aqueous solution

Abstract Acetate esters of six phenolics were formed by the direct addition of 500 μl acetic anydride to 250 ml of a dilute aqueous phenolic solution containing 10 g sodium bicarbonate. In the concentration range of 0.08–0.24 μmoles/1, phenol, o -cresol, m -cresol, p -cresol, 2,4-dichlorophenol and 1-naphthol easily formed acetate esters which provided for improved gas chromatographic characteristics and virtually quantitative recovery from aqueous solution. On extraction with small volumes of methylene chloride, phenol could only be recovered to the extent of 28–41% in the 0.2–2.0 mg/1 range. On the other hand, phenyl acetate, formed in water prior to extraction, was 100% recovered. The stable acetate esters can be analyzed using standard gas chromatographic solumns such as OV-17 or OV-101 while phenolics generally require specially deactivated packings. On 1% SP-1240 DA all of the derivatives, with the exception of m - and p -cresyl acetate, could be separated.

Pharmacokinetics of Ibuprofen Enantiomers in Humans Following Oral Administration of Tablets with Different Absorption Rates

Ibuprofen (IB) is a racemic drug and is administered as such. While activity is due mainly to the S enantiomer, pharmacokinetic interpretations, as well as criteria to assess the bioequivalence of IB formulations, are based on measurements of the total (S + R) drug concentrations. IB enantiomers possess different disposition properties mainly as a result of R-to-S isomeric bioinversion. Inversion is maximal during the absorption phase, suggesting, perhaps, involvement of a presystemic process. This concept was evaluated in healthy subjects by crossover administration of four IB tablets having different absorption rates. The plasma concentrations of the individual isomers were measured using a stereospecific gas chromatographic assay. Differences among the products were insignificant with respect to the extent to the absorption. The S:R concentration ratios rose for 4 to 6 hr and then remained relatively unchanged. This observation was consistent with equal terminal t1/2 values for the enantiomers. There were significant differences between the peak times (Tmax) of the products. The S:R ratios of the concentrations at Tmax of S and AUC also differed; significant positive correlations were found between Tmax and the S:R ratios of Cmax. Thus the extent of R-to-S inversion, and hence the potency of a racemic dose of IB, may be absorption rate dependent.

Simultaneous extraction and quantitation of several bioactive amines in cheese and chocolate

A method is described for simultaneous extraction and quantitation of the amines 2-phenylethylamine, tele-methylhistamine, histamine, tryptamine, m- and p-tyramine, 3-methoxytyramine, 5-hydroxytryptamine, cadaverine, putrescine, spermidine and spermine. This method is based on extractive derivatization of the amines with a perfluoroacylating agent, pentafluorobenzoyl chloride, under basic aqueous conditions. Analysis was done on a gas chromatograph equipped with an electron-capture detector and a capillary column system. The procedure is relatively rapid and provides derivatives with good chromatographic properties. Its application to analysis of the above amines in cheese and chocolate products is described.

Gas chromatographic separation of optically active anti-inflammatory 2-arylpropionic acids using (+)- or (−)-amphetamine as derivatizing reagent

A method is described for the derivatization of several non-steroidal anti-inflammatory arylalkanoic acids (ibuprofen, ketoprofen, naproxen, fenoprofen, flurbiprofen, pirprofen, cicloprofen, tiaprofenic acid, etodolic acid) with optically active amphetamine. The usefulness of this reagent compared to alpha-methylbenzylamine is described. The enantiomers are separated as diastereoisomers using capillary gas chromatography with nitrogen-phosphorus detection. The procedure is readily applied to the quantification of the enantiomers in urine and plasma samples.

Application of a direct aqueous acetylation technique to the gas chromatographic quantitation of nitrophenols and 1-naphthol in environmental water samples

An improved method for the determination of low levels of nitrophenols in aqueous samples has been developed. The method is based on the gas chromatographic analysis of phenols as acetate derivatives which have been prepared directly in water. Recoveries of 97% or greater were obtained from aqueous solutions containing nitrophenols in concentrations ranging from 25 to 100 microgram/l. Concentrations as low as 1 microgram/l were easily detected. Nitrophenols appear to exhibit some resistance to degradation by microorganisms indigenous to the Athabasca River. Water samples to which 139 microgram/l each of o- and p-nitrophenol were added did not show evidence of microbial metabolism over a two week interval. Under identical conditions 100 microgram/l of m-cresol was metabolized within a 3-day period.

Effect of experimental diabetes mellitus and arthritis on the pharmacokinetics of hydroxychloroquine enantiomers in rats

AbstractPurpose. To study the effect of experimental diabetes and arthritis on the pharmacokinetics of hydroxychloroquine (HCQ) enantiomers in rats. Methods. The pharmacokinetic studies were carried out following administration of 40 mg/kg of racemic HCQ to diabetic, insulin-treated diabetic, adjuvant arthritic and control rats. Results. Renal (70% and 62% for R- and S-HCQ, respectively) and non-renal clearance (100% and 145% for R- and S-HCQ, respectively) of HCQ enantiomers were significantly increased in diabetic rats. Diabetes-induced alterations in the disposition of HCQ were reversed by insulin treatment. In arthritic rats, systemic clearance (CL) of HCQ enantiomers was significantly reduced (1.05 ± 0.15 and 1.3 ± 0.19 1/h/kg for R- and S-HCQ, respectively) compared to controls (1.69 ± 0.32 and 1.93 ± 0.34 1/h/kg for R- and S-HCQ, respectively). The fraction unbound of the R- and S-HCQ were 49.4% and 50.5% lower in platelet rich plasma of arthritic rats compared to healthy rats. Increased blood concentrations of HCQ enantiomers in arthritic rats were significantly related to the degree of inflammation. Conclusions. Diabetes significantly increased the CL of both R- and S-HCQ by increasing renal and non-renal clearance. Arthritis caused a significant decrease in CL of HCQ enantiomers through increased binding and a decreased intrinsic clearance. The effect of the diseases on the pharmacokinetics of HCQ, however, was not stereoselective.

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.

Inhibition of insulin metabolism by hydroxychloroquine and its enantiomers in cytosolic fraction of liver homogenates from healthy and diabetic rats.

To elucidate the mechanism by which hydroxychloroquine (HCQ) affects glucose metabolism, the effect of this drug and its enantiomers on insulin metabolism was studied using the cytosolic fraction of liver homogenates from healthy and diabetic rats. Eadie-Hofstee plots were monophasic suggesting that only a one-component enzyme system is involved in insulin degradation in the fraction used. Reaction velocity (V) vs substrate concentration plots were consistent with a Vmax model. HCQ caused a significant reduction in Vmax and Vmax/Km values in both healthy (Vmax, 3.63 +/- 0.46 vs 1.97 +/- 0.13, ng/min/mg; protein P < 0.001; and Vmax/Km 0.265 +/- 0.015 vs 0.112 +/- 0.004, ml/min/g protein) and diabetic rats (Vmax, 0.718 +/- 0.06 vs 0.360 +/- 0.024, ng/min/mg protein; and Vmax/Km, 0.05 +/- 0.002 vs 0.023 +/- 0.001, ml/min/g protein). Significant reduction in the V was observed in the presence of racemic (rac)-, R-, or S-HCQ. Ranking of the inhibitory potency was HCQ > S = R except at highest examined concentration (20 mg/mL) which was HCQ > S > R. In conclusion, the effect of HCQ on insulin degradation appears to be, in part, through inhibition of cytosolic insulin metabolizing enzyme. The effect is not stereoselective except at high concentrations. The R- and S-HCQ may have synergistic effects on inhibition of insulin degradation.

Stereospecific high-performance liquid chromatographic assay of lomefloxacin in human plasma.

This report describes an HPLC assay developed for the quantification of the enantiomers of lomefloxacin (LFLX), a quinolone antibiotic, in plasma. Following addition of racemic acebutolol (internal standard, IS), plasma samples were extracted at pH 7 with a mixture of chloroform-isopentyl alcohol-diethyl ether (71.25:3.75:25, v/v/v). The organic layer was evaporated, and LFLX and IS enantiomers in the resulting residue were derivatized with chloroform solutions of 1% triethylamine and 1% (S)-(+)-(1-naphthyl)ethyl isocyanate, followed by 2% ethyl chloroformate (ECF) 1 min later. Ethanolamine was added 30 s after the addition of ECF. The enantiomers were separated as diastereomers on an 8 x 100 mm Radial Pak normal phase column using a mobile phase of hexane-chloroform-methanol (64.5:33:2.5, v/v/v) pumped at 2.0 ml min-1. The IS was detected by fluorescence at 245 and 420 nm (excitation and emission, respectively) during the first 12 min, after which time the wavelengths were 280 and 470 nm for detection of LFLX. The method: (1) was sensitive and showed excellent linearity (10-1000 ng ml-1, r2 > 0.99) between added enantiomer concentrations and peak-area-ratio (LFLX/IS); and (2) separated LFLX and IS enantiomers within 25 min. The assay is suitable for the quantification of LFLX enantiomers in plasma samples.

Stereospecific high-performance liquid chromatographic assay of acebutolol in human plasma and urine

A sensitive high-performance liquid chromatographic technique is described for the separation of R- and S-acebutolol in human plasma and urine. The procedure involves derivatization with the chiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. The resulting diastereoisomers are quantified using normal-phase high-performance liquid chromatography with fluorescence detection (220/389 nm). Virtual baseline separation, free from interference, with achieved (resolution factor = 1.45). Excellent linearity (r greater than 0.998) was observed throughout the range 10-500 ng/l and 2-100 mg/l in plasma and urine, respectively. Inter-assay variability was less than 5% for each enantiomer at concentrations of 10 ng/ml. This method is applicable for the determination of the pharmacokinetics, in man, of acebutolol enantiomers in plasma and urine.