Validation of a straightforward high performance liquid chromatographic method for morphine quantitation

Kar-Weng Chan

doi:10.1186/s41935-017-0003-0

Abstract

BackgroundMorphine is illegal to use unless it is prescribed by a medical doctor. This compound is commonly abused in the form of illicit heroin.MethodsAn easy-to-use method for determining morphine in illicit heroin using high performance liquid chromatography-photodiode array detector (HPLC-PDA) was developed. With the aid of 50:50 acetonitrile:ammonium formate, this target compound traveling in a pentafluorophenyl (PFP) column was successfully separated from other compounds frequently present in the heroin matrix.ResultsThe method was precise with an intra-day relative standard deviation (RSD) ≤ 0.8% and inter-day RSD < 5%. It was able to detect as low as 0.001 mg/mL morphine. The detector’s response was linear (R2 > 0.999) and reliable for morphine quantitation from 0.005 up to 1 mg/mL. Through recovery studies, accuracy of the method was averagely estimated to be 89.54–101.91%.ConclusionsThis method saves time in terms of mobile phase preparation and cuts cost by excluding additional purchase of expensive chemicals. More importantly, it proves to be able to determine the target analyte with sufficient accuracy and precision.

Highlights

Morphine is illegal to use unless it is prescribed by a medical doctor
Codeine and diacetylmorphine were procured from Toronto Research Chemicals (Canada). 6Monoacetylmorphine was obtained from the Department of Chemistry Malaysia (Malaysia)
High performance liquid chromatography-photodiode array detector (HPLC-PDA) Quantitation of morphine was accomplished with a Waters e2695 HPLC Separations Module coupled with a Waters 2996 Photodiode Array Detector

Summary

Methods

Chemicals and solvents Morphine hydrochloride and caffeine were respectively purchased from Johnson Matthey Macfarlan Smith (Edinburgh) and Merck (Darmstadt). Partial method validation The HPLC method’s selectivity was studied using five illicit heroin samples from different sources and a mixture of five compounds (caffeine, morphine, codeine, 6monoacetylmorphine and diacetylmorphine) frequently present in Malaysian heroin samples (Chan et al 2012). A sample with a low level of morphine (approximately 0.15 mg/mL) and standard solutions. Respectively containing an intermediate level (0.3200 mg/ mL) and high level (0.5186 mg/mL) of morphine were prepared and analyzed repetitively These aliquots were decided as such because the target method will adopt 0.51 mg/mL morphine for calibration and 0.32 mg/mL morphine for performance checking. Serial dilutions at low levels (0.001, 0.002, 0.004 and 0.008 mg/mL morphine) prepared with solutions containing approximately 12 mg/mL caffeine were experimented to study their ideal peak heights and ultraviolet (UV) spectra in order to determine the limit of detection (LOD) for the method. Morphine elutes much earlier, the test with illicit heroin matrices showed that a minimum run time of 18 min (Fig. 1) is mandatory for the all the opiate compounds to purge out to eliminate carry-overs in the subsequent run

Results

Background

Results and discussion

Limitations

Conclusion