UPLC-MS/MS method for determination of retinol and α-tocopherol in serum using a simple sample pretreatment and UniSpray as ionization technique to reduce matrix effects.

Background Our goal was to develop a simple, rapid and precise ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of retinol and α-tocopherol in serum. Currently published LC-MS/MS methods either require complex extraction procedures (liquid-liquid or solid-phase) or do not meet desirable specifications for imprecision in serum (coefficient of variation [CV] <6.8% and 6.9%, respectively). Methods Sample preparation consisted of a simple protein precipitation with ethanol and acetonitrile. Stable isotope-labeled internal standards (IS) and a homemade calibration curve were used for quantification. The analysis was performed using an Acquity I-class Xevo TQ XS LC-MS/MS. Chromatographic runtime was 6.0 min using a reversed phase gradient elution. UniSpray (US) as an ionization technique was compared to electrospray ionization (ESI). Analytical validation included matrix effect, recovery and trueness compared to National Institute of Standards and Technology (NIST) standards and United Kingdom National External Quality Assessment Service (UK NEQAS) samples. Results Intra- and inter-run CVs were <4.9% for retinol and <1.7% for α-tocopherol, both complying with desirable specifications for imprecision. Bias compared to NIST standards was <3.1% for both compounds. The method was linear over the entire tested range. The lower limit of quantification (LLOQ) with US was lower than with ESI for both retinol (0.022 vs. 0.043 mg/L) and α-tocopherol (0.22 vs. 0.87 mg/L). Matrix effects were not significant (<15%) for retinol. However, for α-tocopherol matrix effects of on average 54.0% were noted using ESI, but not with US. Conclusions We developed a fast, precise and accurate UPLC-MS/MS method for the determination of retinol and α-tocopherol in human serum using a single-step sample pretreatment. Ionization using US eliminated the matrix effects for α-tocopherol.

Hb Melusine and Hb Athens-Georgia: potentially underreported in the Belgian population? Four cases demonstrating the lack of detection using common CE-HPLC methods either for glycated hemoglobin (HbA1C) analysis or Hb variant screening.

OBJECTIVE AND IMPORTANCE: Suspected hemoglobin (Hb) variants, detected during HbA1C measurements should be further investigated, determining the extent of the interference with each method. CLINICAL PRESENTATION: This is the first report of Hb Melusine and Hb Athens-Georgia in Caucasian Belgian patients. Intervention & Technique: Since common CE-HPLC methods for HbA1C analysis or Hb variant screening are apparently unable to detect these Hb variants, their presence might be underestimated. HbA1C analysis using CZE, however, alerted for their presence. Moreover, in case of Hb Melusine, even Hb variant screening using CZE was unsuccessful in its detection. CONCLUSION: Fortunately, carriage of Hb Melusine or Hb Athens-Georgia variants has no clinical implications and, as shown in this report, no apparent difference in HbA1C should be expected.

Gentamicin assay in human serum by solid-phase extraction and capillary electrophoresis.

We describe the development of a capillary electrophoresis method for the determination of gentamicin C1, C1a, C2a, and C2 components in human serum. Using a weak cation-exchanger with 20 mM phosphate buffer, pH 7.4, 200 mM borate buffer, pH 9.0, and ammonia/methanol, solid-phase extraction (SPE) of gentamicin components from the human sera was performed. The extract was derivatized with 1,2-phthalic dicarboxaldehyde/mercaptoacetic acid reagent. The derivatives were separated with a background electrolyte comprising 60 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer at pH 9.5 containing 31.6% m/v methanol, and quantified with UV-light absorption detection at 230 nm. The identity of the gentamicin components was confirmed by mass spectrometry. The SPE recovery of the gentamicin ranged from 78% to 93%. The calibration curves were linear from the concentration limit of quantitation (LOQ) to 30 mg/L for the gentamicin mixture. The LOQ for gentamicin C1 was 0.33 mg/L, for C2a 0.23 mg/L, C2 0.25 mg/L, C1a 0.27 mg/L and the concentration limit of detection (LOD) for C1 was 0.15 mg/L, C2a 0.11 mg/L, C2 0.12 mg/L, C1a 0.13 mg/L. Intra-assay relative standard deviation (RSD) values were for C1 (5%), C1a (7%), C2 (6.5%) and C2a (9%); inter-assay RSD values were for C1 (11%), C1a (13.3%), C2 (15%) and C2a (14%). The Pearson's correlation between capillary electrophoresis and immunoassay revealed a linear relationship between these two techniques with r = 0.9. This method for determination of gentamicin C1, C1a, C2a, and C2 in human serum can thus be used in the entire therapeutic concentrations range of gentamicin.

Bioanalysis of tobramycin for therapeutic drug monitoring by solid-phase extraction and capillary zone electrophoresis.

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis (CZE) for the analysis of tobramycin in human serum is presented. An off-line SPE employing a carboxypropyl bonded phase (CBA) cartridge was used for the extraction of tobramycin from human serum. Adsorbed tobramycin was eluted from the CBA cartridge using a mixture of NH(3) (25%, w/v)-methanol (30:70, v/v). After evaporation, the analyte was reconstituted and derivatized with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA). The resulting tobramycin-OPA/MPA derivative was purified, and then identified by mass spectrometry. The tobramycin-OPA/MPA derivative was then analysed by CZE with a background electrolyte (BGE) comprising of 30 mM sodium tetraborate pH 10.0-acetonitrile (ACN) (80:20, v/v) with ultraviolet detection at 230 nm. A linear response was observed in the range of 0.3-30 microg/ml with r(2) = 0.992. The sensitivity of the method was determined by its limit of quantitation (LOQ) and limit of detection (LOD) of 0.3 microg/ml and 0.1 microg/ml, respectively. SPE recovery ranged from 68 to 79% at the trough levels to 98% at the peak levels found in serum. Furosemide has been added as internal standard (IS) to improve precision. For the therapeutic range of tobramycin in serum (2-10 microg/ml) the relative standard deviation (R.S.D.) was less than 11% for the entire SPE/CE process. The method demonstrated excellent selectivity as shown by the lack of interference from a total of 20 drugs investigated. The method was then used in therapeutic drug monitoring of patients receiving the drug.