HPLC with laser-induced native fluorescence detection for morphine and morphine glucuronides from blood after immunoaffinity extraction

Abstract

A new immunoaffinity solid phase extraction of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide is described. An immunoadsorber was applied which was created for the first time by the immobilisation of specific antibodies (polyclonal, host: rabbit) by the sol-gel method. The extraction method in combination with high performance liquid chromatography-fluorescence determination has been validated and shown to be applicable to blood samples of heroin victims in a low concentration range. Blood extracts were essentially free of interfering matrix components when compared to C8-extracts. Additionally, a novel, sensitive and selective detection system for wavelength-resolved analysis of laser-induced fluorescence coupled to HPLC was developed. The analytes were excited with a frequency tripled Ti:Sa laser (lambda=244 nm quasi cw). The total emission spectrum was recorded with a detection system consisting of an imaging spectrograph and a back-illuminated CCD camera. This technique of detection, combined with an extended optical path (at least 6 mm could be illuminated by the laser), resulted in an optimal fluorescence intensity of the analytes. The method permitted the analysis of morphine, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in a low concentration range and could be applied to a complex matrix such as postmortem blood samples because analyte peaks could be discriminated from matrix peaks by their characteristic emission spectra.

Influence of Phenobarbital on Morphine Metabolism and Disposition:LC-MS/MS Determination of Morphine (M) and Morphine-3-Glucuronide (M3G) in Wistar-Kyoto Rat Serum, Bile, and Urine

“…Several methods using various analytical techniques have been utilized for the quantitative analysis of morphine and its glucuronide metabolites in biological matrices. HPLC based methods coupled to UV [25][26][27], fluorescence [28][29][30], electrochemical [29,31], coulometric [27]. Previous methods utilizing radioimmunoassays [32], gas chromatography (GC) [33], and capillary electrophoresis [34,35] have also been used to quantify morphine and its metabolites.…”

Sensitive HPLC–fluorescence detection of morphine labeled with DIB-Cl in rat brain and blood microdialysates and its application to the preliminarily study of the pharmacokinetic interaction between morphine and diclofenac

“…Mor for brain and blood dialysate at S/N ratio of 3 were 0.4 ng/ml (2.7 fmol on column) and 0.6 ng/ml (3.8 fmol on column), respectively. The proposed method could improve the sensitivity of Mor by more than 20 times compared to HPLC-DAD method [7,8] and native FL method [13][14][15]. Furthermore, it was comparable to those of HPLC-ECD [10] and -MS/MS method [20] published recently for determination of Mor in microdialysate.…”

“…Quite recently, HPLC methods combined with tandem MS/MS for very low-level quantification of Mor in biological samples have been described with sub-nanogram per milliliter of quantitation limit [17][18][19][20]. In the HPLC-FL detection methods, either native FL of Mor [13][14][15] or FL of labeled Mor [16] is used. The native FL detection method with 10-50 ng/ml of detection limits is not enough sensitive to detect very small amount of Mor.…”

“…For the determination of Mor, high performance liquid chromatography (HPLC) methods with UV or DAD detection [7,8], electrochemical detection (ECD) [9,10], mass spectrometry (MS) detection [11,12] and fluorescence detection (FL) [13][14][15][16] are widely used. Quite recently, HPLC methods combined with tandem MS/MS for very low-level quantification of Mor in biological samples have been described with sub-nanogram per milliliter of quantitation limit [17][18][19][20].…”

Unveiling Morphine: A Rapid and Selective Fluorescence Sensor for Forensic and Medical Analysis

“…A variety of methods have been developed for the detection of controlled and illicit drugs; they mainly include high-performance liquid chromatography (HPLC) [ 45 , 46 , 47 ], gas chromatography mass spectrometry (GCMS) [ 48 , 49 , 50 ], electrochemical sensors [ 7 , 51 , 52 ], electrochemical luminescence [ 53 , 54 , 55 ], fluorescence spectroscopy [ 56 , 57 ], and surface-enhanced Raman spectroscopy (SERS) [ 58 , 59 , 60 ]. However, these methods have certain shortcomings, such as a complexity in analysis, the need for highly trained operators, long pre-treatment periods, and high costs [ 59 , 61 , 62 ].…”