Comparison of High-Performance Liquid Chromatography and Polyclonal Fluorescence Polarization Immunoassay for the Monitoring of Midazolam in the Plasma of Intensive Care Unit Patients

Abstract

Midazolam (M) is used as an induction agent for anesthesia. The main metabolite is alpha-hydroxymidazolam (OM), which is pharmacologically active. Use of M for sedation is a recent application, rapidly gaining favor. Monitoring of the level of sedation is fundamental in that an excessive and prolonged effect is associated with the risk of complications. Thus, it was felt both necessary and useful to measure circulating M levels. We compared a high-performance liquid chromatography (HPLC) assay with fluorescence polarization immunoassay (FPIA) for the measurement of M in the serum of 138 sedated patients in the intensive care unit (i.e., 179 samples). Response of the OM was also assessed. The degree of crossover of the metabolite was between 76.8 and 32.7%. The equation of the regression line for sigma HPLC (i.e., the sum M + OM) versus FPIA was TDx = 1.1585 sigma HPLC + 143.42 (R = 0.966). The 95% confidence interval for the slope was 1.1551, 1.1619. The regression slope differed significantly from 1 (p < 0.001) and shows that FPIA measurements overestimated concentrations obtained by HPLC on the order of 19%. The discrepancy between the two techniques was all the more notable when concentrations were > 1,000 ng/ml. The relative selectivity of Abbott industrial reagent in terms of benzodiazepines leads to the identification of what might be called a midazolam-like (M-like) activity covering both M and OM. The development of a global FPIA method for measurement of this M-like activity in sedated patients provides a satisfactory solution to the question raised.

Sensitive determination of midazolam and 1′-hydroxymidazolam in plasma by liquid–liquid extraction and column-switching liquid chromatography with ultraviolet absorbance detection and its application for measuring CYP3A activity

“…The limits of detection and quantification were 0.2 and 0.5 ng/ml, respectively, for both midazolam and 1 -hydroxymidazolam. This method is more sensitive than previous reports in which the limits of quantification ranged from 2 to 100 ng/ml [12][13][14][15][16][17][18][19][20][21].…”

“…Several chromatographic methods have been reported for the determination of midazolam and/or its metabolite in plasma [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Gas chromatographic methods appear more sensitive than HPLC; however they require sample derivation for the simultaneous analysis of midazolam and its metabolite [7,8].…”

“…HPLC mass spectrometry-based methods are more sensitive and specific, but the necessary equipment is expensive and not always readily available [9][10][11]. Several HPLC/UV methods have been reported, but the limits of quantification ranged from 2 to 100 ng/ml [12][13][14][15][16][17][18][19][20][21]. More sensitive methods are therefore required for measuring CYP3A activity after clinically safe, low-dose administration of midazolam, because high dose of midazolam intravenously injected has a risk of respiratory depression.…”

Determination of midazolam in human plasma by solid‐phase microextraction and gas chromatography/mass spectrometry

“…45 In most of these applications, a negative or positive midazolam assay may help to screen samples with respect to further and possibly more complex chromatographic analyses. Applications of this method are also feasible for monitoring midazolam plasma concentrations and consequent levels of sedation in intensive care unit patients 46,. 47…”

Quantification of teicoplanin in plasma by LC-MS with online sample clean-up and comparison with QMS® assay

“…To overcome these problems, alternative methods may be used, e.g., chromatography techniques. Previously, teicoplanin was measured by HPLC, usually UV detection [16,[23][24][25]. However, LC-MS is increasingly available for TDM in clinical laboratories, as it offers superior specificity and sensitivity [26].…”