[An LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in human urine and application to urinary excretion study].

ABSTRACT: The study aims to develop an LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in urine samples, and to investigate their urinary excretion characteristics in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection (HTLPI). The urine samples were extracted by methanol, and then separated on a Hedera ODS-2 column with a mobile phase of acetonitrile and 5 mmol · L(-1) ammonium acetate buffer solution containing 0.05% formic acid (20:80). Electrospray ionization source was applied and operated in the positive ion mode using MRM. The method exhibited good linearity over the concentration range of 0.03 -40 µg · mL(-1). The values on both the occasions (intra- and inter-day) were all within 15% at three concentration levels. No matrix effect and carry-over effect were observed. Amygdalin and paeoniflorin were stable in human urine under different storage conditions. Approximately 79.6% of the administered amount of amygdalin was excreted unchanged in urine within 24 h and which was 48.4% for paeoniflorin. The developed LC-MS/MS method can be applied to evaluate the urinary excretion of amygdalin and paeoniflorin.

Identification of amygdalin and its major metabolites in rat urine by LC-MS/MS.

Amygdalin and its metabolites in rat urine were identified using liquid chromatography-electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.

On the metabolism of amygdalin. 1. The LD50 and biochemical changes in rats.

The mean lethal dose (LD50) of amygdalin in rats was found to be 880 mg/kg body weight (BW) by oral administration. However, when 600 mg/kg BW was administered orally with beta-glucosidase, all the rats died. Total and Mg ATPase activities of the heart decreased with increasing levels of administered amygdalin. When 200 mg/kg BW amygdalin was administered 2.3 mg (11.7% of the dose) was excreted intact over 48 h. Amygdalin, 7.4 mg (18.5% of the dose) was excreted when the dose was 400 mg/kg BW, while 7.5 mg (12.4% of the dose) was excreted as intact amygdalin when the dosage was increased to 600 mg/kg BW. Thiocyanate excreted within the same 48-h period was 7.0, 9.1, and 9.5 mumol representing 18, 11.2, and 7.8% of the 200, 400, and 600 mg/kg BW oral dosage, respectively. With 300 mg/kg BW amygdalin administered intraperitoneally, 4.1 mg amygdalin and 3.9 mumol thiocyanate representing 13.7 and 6.5% of the dose, respectively, was excreted. Excretion of intact amygdalin and thiocyanate was uniform when the dose was low (200 mg), but with higher doses over 70% of the excreted products were detected in the urine during the first 24 h.

Determination of amygdalin and its major metabolite prunasin in plasma and urine by high pressure liquid chromatography.

A method is described for the determination of amygdalin and prunasin in plasma ultrafiltrate and urine. Both compounds are separated by high pressure liquid chromatography on a reversed phase column and subsequently detected at 215 nm. The identity of an amygdalin metabolite with prunasin was confirmed by mass spectrometry.

Initial pharmacologic studies of amygdalin (laetrile) in man.

A simple enzymatic assay has been applied to the determination of amygdalin in urine and plasma of patients taking laetrile on their own initiative. Following parenteral administration of laetrile, amygdalin is excreted primarily as the unchanged molecule and urinary recoveries may approach 100 percent. Peak plasma levels after a 6 gm intramuscular dose were 180 microgram/ml. The ratio of amygdalin epimers was unchanged in the urine following parenteral injection.

A simple method for the estimation of amygdalin in the urine.

A procedure for the estimation of D- and D,L-amygdalin in urine is described. Amygdalin is hydrolyzed by beta-glucosidase and base to benzaldehyde, glucose and cyanide. Benzaldehyde is extracted with methylene chloride and the ultraviolet (UV) absorbence determined at 243 nm. The response of human urine "spiked" with amygdalin was linear between 10 and 75 microgram/ml. Mice administered 100 mg/kg of amygdalin intravenously or orally excreted about 70 and 20% of the administered dose, respectively, over 96 hours. In each instance more than 96% of excreted drug equivalents were obtained within the first 24 hours.