Abstract
In this work, a UPLC-MS/MS assay was established for the determination of morphine, codeine, thebaine, papaverine and noscapine in rat plasma. ACQUITY UPLC BEH C18 column was employed for chromatographic separation with the mobile phase comprised acetonitrile-10 mmol L−1 ammonium acetate aqueous solution (0.05% aqueous ammonia) using gradient elution. Midazolam was used as internal standard (IS). Electrospray ionization (ESI) in positive-ion mode with reaction monitoring (MRM) was used for quantitative analysis. The calibration curves for morphine, codeine, thebaine, papaverine and noscapine demonstrated good linearity (r > 0.995) in the range of 5–500 ng mL−1 for morphine and codeine, and 1–100 ng mL−1 for thebaine, papaverine and noscapine. The intra-day and inter-day precisions of morphine, codeine, thebaine, papaverine and noscapine were within 15%, the intra-day and inter-day accuracies were 89–114%, the recovery was better than 65%, and the matrix effects were 96–112%. The developed UPLC-MS/MS assay was successfully applied in the pharmacokinetics of papaverine and noscapine.
Introduction
Pericarpium Papaveris is the dried and mature shell of opium poppy after opium harvest [1, 2]. Morphine, codeine, thebaine, papaverine and noscapine are the main components [3, 4]. These five substances are isoquinoline alkaloids, which have certain medicinal value, but their side effects cannot be underestimated [5, 6]. The alkaloids in the poppy shell can make people drowsy and change their personality, causing a certain degree of contentment and euphoria, which can cause the decline of human attention, thinking and memory performance. Long term consumption will cause mental disorders, hallucinations, and even death due to respiratory arrest. Because the food added with opium poppy shells tastes delicious, it is easy to make consumers addicted, and long-term consumption will produce dependence on it [7]. Therefore, some illegal businesses add illegal raw materials such as opium poppy shells and their water extracts into soup materials or accessories such as hot pot, roast poultry, etc., to attract more diners. Therefore, it is imperative to establish a simple, rapid, and efficient method for the determination of papaver alkaloids.
At present, the detection methods of morphine, thebaine, codeine, papaverine, and noscapine mainly include qualitative identification, thin layer chromatography [8, 9], immunoassay [10], gas chromatography [6], high performance liquid chromatography [11–13], gas chromatography-mass spectrometry [3], and liquid chromatography-tandem quadrupole mass spectrometry [7, 14–16]. Among them, gas chromatography and gas chromatography-mass spectrometry are not suitable for the detection of volatile, strong polar, and pyrolytic compounds, Moreover, the sample needs derivative treatment, which is tedious to operate; enzyme linked immunosorbent assay (ELISA) has cross reaction and poor specificity; the sensitivity of HPLC is low. Bollini et al present a sensitive, specific LC-MS/MS assay for thebaine in urine [15]. Kikura-Hanajiri et al developed a LC-MS/MS with atmospheric pressure chemical ionization (APCI) interface to determinate the urinary excretion of opiates and their metabolites (morphine, codeine, thebaine, noscapine, papaverine, meconic acid and meconin) following inhalation exposure of rats to opium [16]. However, the pharmacokinetics of opium was not reported.
To our best knowledge, there no report of UPLC-MS/MS for simultaneous determination of these five alkaloids in plasma. In this paper, we developed a UPLC-MS/MS assay for determination of morphine, codeine, thebaine, papaverine and noscapine in rat plasma with good selectivity and sensitivity, and then applied to the pharmacokinetics of papaverine and noscapine.
Experimental
Chemicals
Morphine, codeine, thebaine, papaverine, noscapine and midazolam (internal standard (IS)) (all purity ≥98%, Fig. 1) were purchased from Beijing North Weiye Measurement Technology Research Institute (Beijing, China). HPLC-grade acetonitrile and methanol were obtained from Merck Company (Darmstadt, Germany). Ultra-pure water (resistance >18 mΩ) was prepared using a Milli-Q purification system (Bedford, MA, USA).
Chemical structures of morphine (a), codeine (b), thebaine (c), papaverine (d), noscapine (e) and IS (f)
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01108
Instrument conditions
An ACQUITY I-Class UPLC system coupled with a Waters XEVO TQ-S micro triple quadrupole tandem mass spectrometer was for quantitative analysis. A UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) was used for chromatographic separation, and the column temperature was set at 40 °C. The mobile phase consisted of acetonitrile-10 mmol L−1 ammonium acetate aqueous solution (0.05% aqueous ammonia), the flow rate was 0.4 mL min−1. Gradient elution profile was: for 0–0.2 min, acetonitrile kept at 10%; from 0.2 to 1.0 min, acetonitrile from 10% increased into 80%; from 1.0 to 2.6 min, acetonitrile from 80% increased into 90%; from 2.6 to 2.7 min, acetonitrile from 90% changed into 10%; from 2.7 to 4.0 min, acetonitrile kept at 10%.
Nitrogen was used as the cone gas (50 L h−1) and desolvation gas (800 L h−1). The capillary voltage was set to 2.6 kV, the ion source temperature was 145 °C, and the desolvation temperature was 400 °C. It was operated in ESI positive-ion mode, and the quantitation of the five compounds were enabled by operating in MRM by monitoring, Table 1.
Conditions for mass spectrometric detection of five alkaloids
| Compound | Parent ion (m/z) | Daughter ion (m/z) | Cone voltage (v) | Collision voltage (v) |
| Morphine | 286.1 | 165.2* | 30 | 35 |
| 181.2 | 30 | 35 | ||
| Codeine | 300.1 | 165.1 | 30 | 40 |
| 215.1* | 30 | 25 | ||
| Thebaine | 312.1 | 58.1* | 16 | 10 |
| 251.1 | 16 | 15 | ||
| Papaverine | 340.1 | 171.1* | 32 | 30 |
| 202.2 | 30 | 35 | ||
| Noscapine | 414.1 | 220.1* | 30 | 22 |
| 353.3 | 30 | 28 | ||
| IS | 326.2 | 291.4* | 25 | 25 |
| 244.2 | 25 | 25 |
*Quantitative ion.
Standard curve
Stock solutions (5 μg mL−1) of morphine and codeine, (1 μg mL−1) of thebaine, papaverine and noscapine, and (1.0 mg mL−1) of IS were prepared in methanol. The stock solutions were diluted using methanol to obtain working solutions of morphine, codeine, at a range of concentrations (50, 100, 200, 500, 1,000, 2,000, 5,000 ng mL−1), and thebaine, papaverine and noscapine (10, 20, 50, 100, 200, 500, 1,000 ng mL−1).
The morphine, codeine, thebaine, papaverine and noscapine working solutions were added into blank rat plasma to obtain a series concentrations of 5, 10, 20, 50, 100, 200, 500 ng mL−1 for morphine and codeine, and 1, 2, 5, 10, 20, 50, 100 ng mL−1 for thebaine, papaverine and noscapine. Quality control (QC) samples (5, 9, 90 and 450 ng mL−1 for morphine and codeine, 1, 3, 18, and 90 ng mL−1 for thebaine, papaverine and noscapine) were prepared in blank rat plasma under the same conditions.
Sample preparation
Rat plasma (50 μL) was thawed at room temperature, 10 μL of 1 μg mL−1 of the IS added, and 90 μL of acetonitrile-methanol (9:1, v/v) was adjded. Then vortexed for 1.0 min, and centrifuged at 13,000 rpm for 10 min. The supernatant was transferred to a line tube, and 2 μL was injected into the UPLC-MS/MS for analysis.
Method validation
The established method was validated from the aspects of selectivity, standard curve and lower limit of quantification, precision and accuracy, stability, recovery, matrix effect, with reference to the guidelines of the European Union EMA [17].
Selectivity
Six different sources of rat blank plasma and LLOQ samples prepared with corresponding rat blank plasma for UPLC-MS/MS analysis to check whether there was interference in the determination.
Standard curve
The standard curve sample was processed according to the operation under “Sample preparation”. With the concentration of each substance to be measured as the abscissa and the ratio of the peak area of the substance to be measured and the internal standard as the ordinate, the weighted (W = 1/x2) least square method was used for regression calculation. The phase relationship number (r2) of the standard curve obtained was greater than 0.99.
Lower limit of quantification
The lower limit of quantification was to take LLOQ plasma samples for analysis of 6 samples for 3 consecutive days, and calculated the measured concentration of each sample according to the standard curve of the day, and calculated the intra-day and intra-day precision and accuracy, with the signal/noise better than 5. The limit of detection (LOD) of each compound was set as in the signal/noise of 3.
Precision and accuracy
The QC samples was processed according to the operation under “Sample preparation”. Carry out 6 samples analysis for each concentration, and calculated the intra-day and intra-day precision and accuracy respectively within 3 days.
Matrix effect
The matrix effect was 6 blank plasma samples from different sources 50 μL, added 100 μL of acetonitrile-methanol (9:1, v/v), vortex centrifugation, took the supernatant 110 μL. Added working solution 10 μL and internal standard solution 10 μL, and vortex mixing. Took 2 μL to perform UPLC-MS/MS analysis to obtain the corresponding peak areas of the substance to be measured. At the same time, took another deionized water 50 μL instead of blank plasma, treated as above. The matrix effect of analyte was calculated by the peak area ratio of the two treatment methods.
Recovery
Took the quality control samples, and operated according to the “Sample preparation”. Six samples are analyzed for each concentration (A). At the same time, took another blank plasma 50 μL. Added acetonitrile-methanol (9:1, v/v) 100 μL, vortex centrifugation. Took the supernatant 110 μL. Added working solution 10 μL and internal standard solution 10 μL. Vortex mixing and conduct six samples analysis for each concentration (B). Took2 μ L for LC-MS/MS analysis, the recovery was calculated based on the peak area ratio of the two treatment methods for each concentration, and the calculation formula was A/B × 100%.
Stability
The stability of morphine, codeine, thebaine, papaverine and noscapine at low, medium and high concentration plasma samples was investigated. The plasma samples were placed at room temperature for 24 h, after precipitation protein treatment at room temperature for 24 h, and after treatment at 4 °C for 24 h. The plasma samples were subjected to three freeze-thaw cycles at −20 °C, and the stability of long-term storage at −20 °C.
Pharmacokinetics
Sprague Dawley (SD) rats (male, 200–220 g) were from the Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China). All experimental procedures and protocols were approved by the Animal Care Committee of Wenzhou Medical University. Twelve rats were administered papaverine, intravenous (iv, 1 mg kg−1) and oral (po, 10 mg kg−1), six rats for each dosage. Another twelve rats were administered noscapine, intravenous (iv, 1 mg kg−1) and oral (po, 10 mg kg−1), six rats for each dosage. At 0.0833, 0.25, 1, 2, 4, 6, 8, 12 h (for papaverine) and 0.0833, 0.25, 1, 2, 4, 6, 8, 12, 24 h (for noscapine) post-administration, from the caudal vein, 0.2 mL of blood was collected into a 1.5 mL heparinized tube, and centrifuged at 13,000 rpm for 10 min. Then, 50 μL of the plasma was obtained to a 1.5 mL tube and stored at −20 °C until analysis. The main pharmacokinetic parameters were fitted by the DAS 2.0 software (Shanghai University of traditional Chinese Medicine).
Results and discussion
Method development
Comparing the ESI positive and negative modes, morphine, codeine, thebaine, papaverine and noscapine were best-suited for detection in ESI positive-ion mode. Morphine, codeine, thebaine, papaverine and noscapine were prepared in rat blank plasma at a concentration of 50 ng mL−1. Methanol-acetonitrile (1:9, v/v), methanol-acetonitrile (1:1, v/v), acetonitrile, and 10% trichloroacetic acid in methanol were tried, and it was found that methanol-acetonitrile (1:9, v/v) had the best extraction recovery.
Chromatographic separation conditions influenced retention time and peak shape [18, 19]. We investigated different HSS T3 and BEH C18 columns combined with various mobile phases (acetonitrile-0.1% formic acid, acetonitrile-10 mmol L−1 ammonium acetate aqueous solution, acetonitrile-10 mmol L−1 ammonium acetate aqueous solution (0.1% formic acid), acetonitrile-10 mmol L−1 ammonium acetate aqueous solution (0.05% aqueous ammonia), methanol-10 mmol L−1 ammonium acetate aqueous solution). When 0.1% formic acid aqueous solution-acetonitrile was used as mobile phase, the peak shape becomes wider, the separation between components was poor, and morphine was not retained in the chromatographic column. When different ammonium acetate systems are used as mobile phases, with the increase of pH value, the separation degree of the five components increases, and the retention time increases, and found that BEH C18 and an acetonitrile-10 mmol L−1 ammonium acetate aqueous solution (0.05% aqueous ammonia) displayed symmetry and sharpness chromatographic peaks. BEH C18 was better than HSS T3 for the analysis of the broad range of compounds and has excellent tolerance to extreme pH condition. The extreme pH condition for BEH C18 was between 1 and 12, while it was between 1 and 8 for HSS T3. Acetonitrile-10 mmol L−1 ammonium acetate aqueous solution (0.05% aqueous ammonia) was weak alkaline, and it was more suitable for BEH C18 column.
The elution mode in this work adopted rapid gradient elution. Gradient elution had a relatively low initial organic phase, which could enhance the chromatographic retention of compounds. On the other hand, the cleaning of high organic phase could prevent the accumulation of endogenous matrix introduced by protein precipitation from producing matrix effect. In addition, high organic phase elution could improve the atomization efficiency and significantly improve the mass response. The chromatographic operation time of this method was relatively short, only 4 min, and the retention time of each compound was between 1.6 and 2.2 min, which was relatively suitable.
Selectivity
The retention time of morphine, codeine, thebaine, papaverine, noscapine, and the IS were 1.62, 1.81, 2.04, 2.11, 2.36 and 2.22 min in Fig. 2, respectively, and no interference was found.
UPLC-MS/MS of morphine, codeine, thebaine, papaverine, noscapine and IS in rat plasma, (a) blank plasma, (b) blank plasma spiked with morphine, codeine, thebaine, papaverine, noscapine and IS
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01108
Standard curve
The calibration curves of morphine, codeine, thebaine, papaverine and noscapine in rat plasma demonstrated good linearity, Table 2. The LLOQ of morphine and codeine in rat plasma were 5 ng mL−1, and thebaine, papaverine and noscapine in rat plasma were 1 ng mL−1. The LOD of morphine and codeine in rat plasma were 1.5 ng mL−1, and thebaine, papaverine and noscapine in rat plasma were 0.3 ng mL−1.
Accuracy, precision, matrix effect, and recovery of five alkaloids in the rat plasma (n = 6)
| Compound | Concentration (ng mL−1) | Precision (RSD%) | Accuracy (%) | Recovery (%) | Matrix effect (%) | ||
| Intra-day | Inter-day | Intra-day | Inter-day | ||||
| 5 | 15.0 | 14.1 | 111.7 | 89.5 | 70.8 | 105.4 | |
| Morphine | 9 | 10.8 | 7.7 | 103.0 | 97.8 | 68.8 | 108.4 |
| 90 | 8.7 | 5.6 | 89.8 | 99.9 | 77.0 | 96.9 | |
| 450 | 11.5 | 9.0 | 103.0 | 101.5 | 71.1 | 105.0 | |
| 5 | 8.2 | 9.1 | 108.1 | 96.9 | 78.5 | 107.3 | |
| Codeine | 9 | 10.0 | 7.2 | 98.7 | 92.7 | 81.0 | 99.6 |
| 90 | 8.5 | 6.7 | 100.2 | 102.7 | 85.6 | 96.6 | |
| 450 | 7.5 | 7.4 | 106.2 | 102.1 | 86.1 | 108.8 | |
| 1 | 8.5 | 8.7 | 107.1 | 105.5 | 72.4 | 111.0 | |
| Thebaine | 3 | 12.2 | 5.5 | 99.1 | 91.5 | 76.4 | 105.3 |
| 18 | 6.9 | 6.3 | 103.2 | 101.4 | 83.5 | 96.3 | |
| 90 | 8.2 | 8.3 | 101.3 | 105.9 | 79.0 | 101.8 | |
| 1 | 11.9 | 14.1 | 96.5 | 113.5 | 67.0 | 105.8 | |
| Papaverine | 3 | 11.7 | 12.1 | 104.6 | 106.0 | 65.2 | 108.7 |
| 18 | 9.8 | 7.7 | 91.8 | 91.0 | 68.2 | 103.9 | |
| 90 | 8.2 | 5.3 | 99.5 | 98.4 | 66.6 | 106.4 | |
| 1 | 12.8 | 10.1 | 100.6 | 113.5 | 74.0 | 111.1 | |
| Noscapine | 3 | 9.0 | 5.9 | 99.0 | 100.0 | 72.9 | 105.0 |
| 18 | 6.4 | 5.7 | 95.3 | 95.1 | 73.3 | 104.4 | |
| 90 | 8.2 | 8.1 | 94.0 | 97.6 | 72.0 | 106.4 | |
Precision, accuracy, recovery and matrix effects
The intra-day and inter-day precisions of morphine, codeine, thebaine, papaverine and noscapine were within 15%, the intra-day and inter-day accuracies were 89–114%, the recovery was better than 65%, and the matrix effects were 96–112% (Table 3).
Regression equation, LLOQ and LOD of five alkaloids in the rat plasma
| Compound | Regression equation | Concentration range (ng mL−1) | r | LLOQ (ng mL−1) | LOD (ng mL−1) |
| Morphine | y = 0.0005x−0.0055 | 5–500 | 0.9974 | 5 | 1.5 |
| Codeine | y = 0.0014x−0.0108 | 5–500 | 0.9958 | 5 | 1.5 |
| Thebaine | y = 0.0112x−0.0122 | 1–100 | 0.9994 | 1 | 0.3 |
| Papaverine | y = 0.0017x−0.0036 | 1–100 | 0.9983 | 1 | 0.3 |
| Noscapine | y = 0.0058x−0.0048 | 1–100 | 0.9995 | 1 | 0.3 |
Wherein x represented the concentration of compound in the plasma, and y represented the ratio of the peak area of compound to the IS.
Stability
The accuracy of morphine, codeine, thebaine, papaverine and noscapine was between 90% and 112%, and the RSD was within 15%. These results indicated that five compound had good stability.
Pharmacokinetics
Papaverine is mainly used to relieve cerebral and peripheral vascular diseases with arterial spasm, and to treat cerebral thrombosis, pulmonary embolism, acrospasm and arterial thrombotic pain [20, 21]. It can also be used for the treatment of intestinal, ureteral, and biliary spasm pain and dysmenorrhea, and as a component of the compound bronchodilator spray. And it can also be used for hypertension, angina pectoris, and cardiac ischemia with arrhythmia. When used in large doses, it can cause serious arrhythmia and even death. Noscopine, an antitussive drug, has similar antitussive effect to codeine and is not addictive [22]. It is suitable for patients with irritating dry cough. There are few adverse reactions, but large doses may stimulate breathing and cause bronchospasm, so the prescribed dosage should be strictly followed. Therefore, it is of great clinical significance to establish a method for five alkaloids in plasma.
The non-compartmental model was used to fit the main pharmacokinetic parameters, Table 4. The concentration-time curves for papaverine and noscapine in rat plasma are shown in Fig. 3. The half-lives (t1/2z) of papaverine and noscapine in the rats after intravenous administration were 1.0 ± 0.2 h, 6.2 ± 2.2 h, respectively, while the t1/2z after oral administration were 2.4 ± 0.6 h, 1.6 ± 0.5 h, respectively, indicated that they were metabolized very fast. The papaverine and noscapine had very low oral bioavailabilities of 0.18% and 1.32%, respectively.
Main pharmacokinetic parameters after intravenous (iv) and oral (po) administration of papaverine and noscapine in rats
| Parameters | Unit | Papaverine | Noscapine | ||
| po (10 mg kg−1) | iv (1 mg kg−1) | po (10 mg kg−1) | iv (1 mg kg−1) | ||
| AUC(0-t) | ng mL−1*h | 41.8 ± 7.7 | 2301.1 ± 388.6 | 184.2 ± 71.1 | 1394.6 ± 256.1 |
| AUC(0-∞) | ng mL−1*h | 44.4 ± 8.2 | 2301.6 ± 388.8 | 191.7 ± 79.1 | 1455.2 ± 234.3 |
| MRT(0-t) | h | 1.8 ± 0.4 | 1.3 ± 0.1 | 1.5 ± 0.5 | 4.6 ± 0.5 |
| MRT(0-∞) | h | 2.4 ± 0.4 | 1.4 ± 0.1 | 1.7 ± 0.6 | 6.2 ± 1.2 |
| t1/2z | h | 2.4 ± 0.6 | 1.0 ± 0.2 | 1.6 ± 0.5 | 6.2 ± 2.2 |
| Tmax | h | 0.3 | 0.1 | 0.3 ± 0.1 | 0.1 |
| CLz/F | L h−1kg | 230.7 ± 39.5 | 0.4 ± 0.1 | 58.9 ± 22.9 | 0.7 ± 0.1 |
| Vz/F | L kg−1 | 807.7 ± 262.9 | 0.6 ± 0.2 | 132.6 ± 73.2 | 6.4 ± 2.8 |
| Cmax | ng mL−1 | 35.9 ± 6.3 | 1762.1 ± 309.5 | 159.7 ± 31.7 | 553.0 ± 51.4 |
Plasma concentration-time curves after intravenous (iv, 1 mg kg−1) and oral (po, 10 mg kg−1) administration of papaverine, noscapine in rats
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01108
Conclusion
In this study, we established a method for quantitative analysis of 5 kinds of papaverine alkaloids, such as morphine, codeine, thebaine, papaverine, and nacodine, with the LOD of 0.3–1 ng mL−1, LLOQ of 1–5 ng mL−1. The linear relationship of the method was good, and the correlation coefficient was greater than 0.995. This method was accurate, reliable, sensitive, and simple for pretreatment. It can be applied to the detection of papaver alkaloids in plasma.
Conflict of interest statement
The author(s) declare(s) that there is no conflict of interest regarding the publication of this paper.
Data availability statement
The data used to support the findings of this study are included within the article.
Acknowledgements
This work was supported by the Ningbo Natural Science Foundation (202003N4265), Basic Public Welfare Research Project of Zhejiang Province (LGD20H090005), Medical Science and Technology Project of Zhejiang Province (2022RC293).
Reference
- 3.↑
Tittarelli, R.; Gismondi, A.; Di Marco, G.; Mineo, F.; Vernich, F.; Russo, C.; Marsella, L. T.; Canini, A. Biology (Basel) 2022, 11.
- 5.↑
Zhang, H.; Shi, X.; Qiu, G.; Wang, X.; Zhang, C.; Zhang, X.; Wu, F.; Xu, X.; Li, C.; Pan, X. Se Pu 2020, 38, 861–867.
- 11.↑
Bagheri, M.; Taheri, M.; Farhadpour, M.; Rezadoost, H.; Ghassempour, A.; Aboul-Enein, H. Y. J. Chromatogr. A. 2017, 1511, 77–84.
- 12.
Li, B.; Petersen, N. J.; Payan, M. D.; Hansen, S. H.; Pedersen-Bjergaard, S. Talanta 2014, 120, 224–229.
- 13.
Shamsipur, M.; Fattahi, N. J. Chromatogr. B Analyt Technol. Biomed. Life Sci. 2011, 879, 2978–2983.
- 16.↑
Kikura-Hanajiri, R.; Kaniwa, N.; Ishibashi, M.; Makino, Y.; Kojima, S. J. Chromatogr. B Analyt Technol. Biomed. Life Sci. 2003, 789, 139–150.
- 17.↑
Agency, E. M. London: European Medicines Agency, 2011. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf.
- 21.↑
Badea, I. A.; Vladescu, L.; David, I. G.; David, V.; Litescu, S. C. Anal. Lett. 2010, 43, 1217–1229.
