Colette Fierens

The qualitative and quantitative determination of quinolones of first and second generation by capillary electrophoresis.

Capillary electrophoresis (CE) was applied to the study of 10 quinolones of first and second generation--nalidixic acid, oxolinic acid, pipemidic acid, cinoxacin, norfloxacin, ciprofloxacin, ofloxacin, pefloxacin, fleroxacin, and flumequine. Separation was performed on a fused silica capillary (75 microm-60 cm) using a phosphate buffer (pH 7.0, 125 mM). Detection was at 214 nm. Only norfloxacin and ciprofloxacin cannot be separated in this way. Because of the specificity of the method, the identification of the individual quinolones by their migration time was possible. The same system has been applied for the quantitative determination of quinolones in tablets and capsules. Excipients do not adversely affect the results. Some parameters (linearity, precision, accuracy) were validated. Especially the possibility of simultaneous quantification and identification of the active ingredient in the finished product is very attractive.

Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard.

We describe the first results of a quantitative LC-tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was approximately 0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of approximately 75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.

Isotope dilution-gas chromatography/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of triiodo- L-thyronine in serum

Isotope dilution-gas chromatography/mass spectrometry (ID-GC/MS) and isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) methods have been developed for an determination of triiodothyronine (T3) in serum and their potential as candidate reference methods investigated. In both methods, (13)C(9)-T3 was used as internal standard. Sample pretreatment consisted of deproteinization, extraction and high performance liquid chromatography (HPLC) purification. Conversion of serum thyroxine (T4) to T3 was controlled by adding (13)C(6)-T4. For GC/MS, T3 and (13)C(9)-T3 were converted to the N,O-di-heptafluorobutyryl (HFB) methyl ester derivatives and monitored at m/z 844 and 853. For LC/MS with electrospray ionization, the transitions m/z 652/661 to 606/614 were monitored. For use of the methods as candidate reference methods, special attention was paid to the calibration and the measurement protocol (duplicate analysis of each sample on three occasions). Evaluation of the ID-GC/MS and ID-LC/MS/MS methods showed the absence of interference by reverse T3 and T4, a limit of detection of 100 pg (GC/MS) and 18 pg (LC/MS), a recovery of 100 +/- 1.5% (95% confidence interval) and good precision (the total coefficient of variation, n = 6, was typically 1.5%). In addition to the recovery study, the accuracy of the methods was proven by method comparison (ID-GC/MS vs. LC/MS/MS) on three sera, showing a maximum deviation of 1.1%. Finally, the ID-GC/MS method was applied for measurement of 10 different human sera with a T3 concentration range from 0.6 to 7.3 ng/mL.

Standardization of C-Peptide Measurements in Urine by Method Comparison with Isotope-Dilution Mass Spectrometry

In the past, standardization of measurements of diagnostically important polypeptides and proteins was hampered by the noncommutability of primary standards (1). One way to overcome this problem is to establish a method comparison with a reference measurement procedure. Until now, reference measurement procedures, such as isotope-dilution mass spectrometry (ID-MS), have been scarce. Recent developments in the MS field, however, have made the technique easily applicable to the analysis of polypeptides and proteins (kinetic studies, sequence analysis, and determination of molecular mass and posttranslational modifications). To the best of our knowledge, only two groups have used ID-MS for the quantitative determination of a specific polypeptide/protein. One of these groups described the measurement of apolipoprotein A-I after enzymatic digestion (2), the other described offline ID-liquid chromatography (LC)-MS assays for serum proinsulin, insulin, and C-peptide (3)(4). Neither group, however, examined the potential of ID-MS for standardization of the respective routine test systems (usually, immunoassays). Here we report on the use of an ID-MS measurement procedure for standardization/recalibration of C-peptide measurements in urine by use of a method-comparison study with split-sample measurements. In view of the model character of the study for future applications, we chose urinary C-peptide over the clinically more important serum C-peptide because of the ease of sample collection and MS measurement. The measurement procedure applies online ID-LC-electrospray tandem MS (ID-LC-MS/MS) and is described in detail elsewhere (5). For calibration, it makes use of a commercial C-peptide preparation with a peptide content of 89% and a purity by HPLC of >99% (according to the manufacturer’s information). This purity was taken into account for calculation of the C-peptide content in the calibrators. The C-peptide preparation was delivered in a vial containing 250 μg of freeze-dried material; a calibration solution was prepared by carefully weighing the added volume (∼1 mL) of …

Application of a C-peptide electrospray ionization-isotope dilution-liquid chromatography-tandem mass spectrometry measurement procedure for the evaluation of five C-peptide immunoassays for urine.

This study applied electrospray ionization-isotope dilution-liquid chromatography-tandem mass spectrometry for the evaluation of five urinary C-peptide immunoassays via split-sample measurements. The immunoassays measured in duplicate in the same run, the comparison method in triplicate over different runs. From the data, the within-run imprecision and the method comparison total RSDs were calculated. Regression analysis revealed on the one hand systematic differences, on the other, an excellent correlation between the test and comparison methods. From the spread of the data around the regression line in comparison with the 95% prediction intervals from the total RSD, sample-related effects and/or specificity problems were apparent and investigated.