A sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the determination of morphine and codeine in human urine. The GC-MS conditions were developed. The analysis was carried out on a HP-1MS column (30 m × 0.25 mm, 0.25
Morphine and codeine are naturally occurring alkaloids in opioid plants, have long been used as a drug, and are also abused. While the presence of illicit drugs or their metabolites in urine is an evidence of intake, their concentrations in blood are expected to correlate with their effects on the central nervous system [
There are numerous papers published about the simultaneous determination of Morphine and Codeine in human fluids, including the micellar electrokinetic chromatography (MEKC) method [
Currently, urine sampling has been extensively employed for the evaluation of drug consumption. Although through in saliva is another approach; the reliability of saliva analysis is limited by the fact that analyte levels, and even the availability of required sample volume, are again dependent on several physiological factors, nutrition and fluid intake, while the biological effects of the consumed illicit substance may also be a significant factor [
Sample preparation is a key step for the determination of drugs in biological samples. The simple and effective ethyl acetate extraction was employed in our work, and ethyl acetate was adopted because of its high extraction efficiency. Pyridine is a catalytic solvent for reactions with propionic anhydride. Propionic anhydride was chosen as the derivatization reagent because it exhibited better effect than acetic anhydride or trifluoroacetic acid anhydride, which could provide preferable stability, and the disadvantage of acetyl derivatives indistinguishable from morphine and the 6-AM can be avoided. Kushnir et al. [
Morphine [10
Analysis was performed on an Agilent 6890N gas chromatograph (GC) coupled with an Agilent 5975B mass spectrometer (MS, Agilent Technologies, Wilmington, DE, USA). Samples were injected using an Agilent autosampler unit.
The capillary column used was a HP-1MS [30 m × 0.25 mm, 0.25
Mass spectra of morphine (a) and codeine (b) in SIM mode with EI (+) source.
The primary standard stock solutions of morphine (100
Specificity was determined by analysis of blank urine, without addition of morphine and codeine to determine possible interference with these compounds.
To evaluate the linearity, the calibration curves were generated using the analyte peak area by linear regression on three consecutive days. The LLOQ was estimated in the process of calibration curve construction and was defined as the lowest concentration for which precision (RSD) was better than 20%.
QC samples at three concentration levels (50, 200, and 1600 ng/mL for morphine and codeine) were analyzed to assess the accuracy and precision of the method. Again, the assays were performed on three separate days, and on each day six replicates of the QC samples at each concentration level were analyzed. The assay accuracy was calculated as relative error. The assay precision for each QC level was determined as the relative standard deviation (RSD) of the measured concentrations. The intra- and interday precisions were required to be below 15%, and the accuracy was required to be within ±15%.
Stability in urine was assessed in the autosampler at room temperature for 12 h. The effect of three freeze-thaw cycles was also investigated.
Figure
Chromatograph of urine sample containing 1600 ng/mL morphine (a) and codeine (b) processed through the procedure.
Calibration curves for morphine and codeine were generated by linear regression of peak area ratios against concentrations, respectively. The regression equation for the calibration plot were
The LLOQ for morphine in human urine was 25 ng/mL and the precision and accuracy at LLOQ were 10.5% and 87.6%, respectively. The LLOQ for codeine in human urine was 25 ng/mL and the precision and accuracy at LLOQ were 13.8% and 88.9%, respectively.
The precision of the method was determined by calculating RSD for QCs at three concentration levels over three validation days. Intraday precision was 12% or less and the interday precision was 13% or less at each QC level. The accuracy of the method ranged from 87.2% to 99.7% at each QC level. Assay performance data are presented in Table
The results of precision, accuracy, and recovery studies for morphine and codeine in human urine
Compound | Concentration | RSD (%) | RE (%) | Recovery (%) | ||
---|---|---|---|---|---|---|
(ng/mL) | Intraday | Interday | Intraday | Interday | ||
Morphine | 50 | 4.3 | 8.2 | −0.3 | −6.5 | 86.1 |
200 | 4.4 | 7.4 | −5.2 | −10.1 | 75.5 | |
1600 | 5.3 | 9.3 | −6.9 | 5.7 | 80.2 | |
| ||||||
Codeine | 50 | 10.5 | 13.0 | 8.5 | −7.3 | 78.9 |
200 | 11.4 | 9.8 | −3.9 | −12.8 | 79.7 | |
1600 | 5.2 | 12.9 | −9.9 | −8.2 | 86.8 |
All the stability studies of morphine and codeine in human urine were conducted at three concentration levels (50, 200, and 1600 ng/mL for morphine and codeine) with three replicates for each concentration. The stability results showed that morphine and codeine in human urine were stable during three freeze-thaw cycles. Stability of morphine and codeine extracts in the sample solvent on autosampler was also observed over a 12 h period. The results of stability experiments are listed in Table
Summary of stability of morphine and codeine under various storage conditions
Compound | Condition | Concentration (ng/mL) | RSD | RE |
---|---|---|---|---|
Added | (%) | (%) | ||
Morphine | Three freeze–thaw cycles | 50 | 2.5 | −10.1 |
200 | 1.4 | −8.9 | ||
1600 | 4.3 | −12.5 | ||
Autosampler ambient 12 h | 50 | 2.1 | −10.5 | |
200 | 1.3 | −6.2 | ||
1600 | 3.5 | −9.2 | ||
| ||||
Codeine | Three freeze–thaw cycles | 50 | 3.2 | −8.9 |
200 | 3.3 | −11.2 | ||
1600 | 3.0 | −14.1 | ||
Autosampler ambient 12 h | 50 | 3.3 | −11.1 | |
200 | 3.2 | −12.9 | ||
1600 | 3.3 | −14.4 |
A stable, selective, and sensitive GC-MS method has been developed for the simultaneous determination of codeine and its metabolite morphine in human urine. This developed method with derivatization for sample preparation was successfully applied for the determination of morphine and codeine in human urine for methodological study.
The authors declare that they have no conflict of interests.
This work was supported by the fund of the Youth Talent Program Foundation of The First Affiliated Hospital of Wenzhou Medical College (qnyc010). The authors thank Youting Zhang for valuable discussion and assistance with the modification. Furthhermore, the authors acknowledge the editors and the anonymous reviewers for their insightful suggestions on this work.