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Comparison of Capillary Electrophoresis with HPLC for Diagnosis of Factitious Hypoglycemia

¹ Laboratory of Separative Techniques, IRCCS Ospedale San Raffaele, 20132 Milano, Italy

² Unit of Metabolic Disease, Azienda Ospedaliera e Universita’, 35100 Padova, Italy.

³ Endocrinology and Diabetes Unit, Istituto Clinico Humanitas, 20089 Rózzano, Milano, Italy.

⁴ Institute of Endocrinological Science, IRCCS Ospedale Maggiore, 20122 Milano, Italy.
^a Address correspondence to this author at: Laboratory of Separative Techniques, Laboratory Analysis, H. San Raffaele, via Olgettina 60, 20132 Milano, Italy. Fax 39-02-26432640; e-mail paroni.rita{at}hsr.it

Background: The diagnosis of "factitious hypoglycemia" is essentially based on the disclosure of hypoglycemic agents in blood or urine. The aim of this study was to evaluate the performance of capillary electrophoresis (CE) as a quantitative method for determination of chlorpropamide, tolbutamide, glipizide, gliclazide, and glibenclamide in serum.

Methods: Serum samples (1 mL), with internal standard added, were purified by solid-phase extraction on OASIS^TM HLB cartridges (Waters), dried under reduced pressure, and reconstituted with 30–60 µL of acetonitrile:H₂O. Analysis was carried out by micellar electrokinetic capillary chromatography in 5 mmol/L borate, 5 mmol/L phosphate, 75 mmol/L sodium cholate, pH 8.5, containing 25 mL/L methanol. Separation was accomplished in a 20 cm x 50 µm (i.d.) silica capillary at 25 °C and a constant voltage of +10 kV. Pharmacokinetics of gliclazide (80-mg tablet) in a diabetic patient were assayed by both HPLC and CE. Two hypoglycemic patients positive by HPLC analysis for unreported gliclazide and tolbutamide overdose were also screened by CE.

Results: Separation of six drugs (including the internal standard) was accomplished in 5 min plus 5 min rinsing. The between-day CV of the ratio of the areas of the sulfonylurea drugs to internal standard was <1% (n = 10). Linearity (r² 0.998) and recovery (80%) were good for all sulfonylurea drugs tested. Pharmacokinetic curves for gliclazide by CE and HPLC were superimposable. CE analysis confirmed the HPLC diagnosis of surreptitious abuse of gliclazide and tolbutamide.

Conclusion: CE is a useful tool in the clinical chemistry and toxicology laboratory for drug monitoring and pharmacokinetic investigations.

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