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Journal of Pharmaceutical and Biomedical Analysis 180 (2020) 113042
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Determination of buprenorphine, naloxone and phase I and phase II metabolites in rat whole blood by LC–MS/MS Camille Cohier a,b,c,d , Sophie Salle d , Anne Fontova d , Bruno Mégarbane a,b,c,e,∗ , Olivier Roussel a,b,c,d a
Inserm, U1144, Paris, France Paris-Descartes University, UMR-S 1144, Paris, France c Paris-Diderot University, UMR-S 1144, Paris, France d Forensic Toxicology Unit, Forensic Sciences Institute of the French Gendarmerie, Pontoise, France e Department of Medical and Toxicological Critical Care, Lariboisière Hospital, Paris, France b
a r t i c l e
i n f o
Article history: Received 7 August 2019 Received in revised form 9 December 2019 Accepted 9 December 2019 Available online 16 December 2019 Keywords: Buprenorphine Naloxone Glucuronide Whole blood Protein precipitation LC–MS/MS
a b s t r a c t Buprenorphine and buprenorphine/naloxone combination are maintenance treatments used worldwide. However, since their marketing, despite ceiling respiratory effects, poisonings and fatalities have been attributed to buprenorphine misuse and overdose. Therefore, to better understand the mechanisms of buprenorphine-related toxicity in vivo, experimental investigations have been conducted, mainly in the rat. We developed a liquid chromatographic-tandem mass spectrometric (LC–MS/MS) method with electrospray ionization for the simultaneous quantification of buprenorphine, naloxone and their metabolites (norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide and naloxone glucuronide) in rat whole blood. Compounds were extracted from whole blood by protein precipitation and chromatographically separated using gradient elution of aqueous ammonium formate and methanol in a Raptor Biphenyl core-shell column (100 mm x 3,0 mm x 2,7 m). Following electrospray ionization, quantification was carried out in the multiple reaction monitoring (MRM) mode by the tandem mass spectrometer API 3200 system. The LC-MS/MS method was validated according to the currently accepted criteria for bioanalytical method validation. The method required small sample volumes (50 L) and was sensitive with limits of quantification of 6.9, 6.2, 3.6, 3.3, 1.3 and 57.7 ng/mL for buprenorphine, norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide, naloxone and naloxone glucuronide respectively. The upper limit of quantification was 4000 ng/ml for all the studied compounds. Trueness (88–115 %), repeatability and intermediate precision (both <15%) were in accordance with the international recommendations. The procedure was successfully used to quantify these compounds in the whole blood sample from one rat 24 h after the intravenous administration of buprenorphine/naloxone (30.0/7.5 mg/kg). © 2019 Elsevier B.V. All rights reserved.
1. Introduction Buprenorphine (BUP), a semi-synthetic opioid derivate of thebaine, is widely used as an effective maintenance treatment in heroin addicts. Since 2006, a BUP/naloxone (BUP/NLX) combination has been marketed with the aim of preventing intravenous misuse of crushed buprenorphine tablets. In humans [1–3] as well as rodents [4,5], BUP was shown to exhibit ceiling respiratory effects in contrast to other opioids, supporting its safety profile. However,
∗ Corresponding author at: Department of Medical and Toxicological Critical Care, Lariboisière Hospital, 2 Rue Ambroise Paré 75010, Paris, France. E-mail address: [email protected] (B. Mégarbane). https://doi.org/10.1016/j.jpba.2019.113042 0731-7085/© 2019 Elsevier B.V. All rights reserved.
reports emerged of BUP-associated poisonings with typical opioid features and respiratory depression and even fatalities [6–8] while toxicity prevention using the BUP/NLX combination remains a matter of debate [9,10]. BUP is highly bound to blood proteins (96%) and metabolized in the liver by the cytochrome P450 3A4 into the active and toxic metabolite norbuprenorphine (NorBUP) [11–13]. Both compounds are extensively conjugated by UDP-glucuronyl transferase to form the active metabolites, BUP-glucuronide (BUP-GLUC) and NorBUP-glucuronide (NorBUP-GLUC) [14]. The mu-opioid receptor antagonist NLX is also conjugated to form an active metabolite, NLX-glucuronide (NLX-GLUC) [15]. In autopsy cases where BUP or NorBUP had been detected in blood or urine, differences in blood concentrations of BUP and NBUP
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between BUP-attributed fatalities and cases with other causes of death were not clinically relevant [7,8,10,16], suggesting that BUP overdose cannot explain the patient’s death by itself in the majority of cases. Mechanisms of BUP-related toxicity remain thus still unclear, requiring extensive investigations based on experimental studies, which mainly involve rats. Due to the small blood volumes sampled from rats, quantification of BUP and its metabolites in whole blood is requested to facilitate the experimental procedures and limit the number of animals in pharmacokinetic studies. Quantification in whole blood is challenging due to the complexity and diversity of the components included in such a matrix, additionally requiring a sensitive and specific detection system such as liquid chromatography coupled to tandem mass spectrometry (LC MS/MS). LC–MS/MS is considered as the technique of reference for drug analysis including BUP in forensic toxicology and situations requiring multiple sampling like pharmacokinetic studies [14,17–20]. Various sample preparation techniques were developed using solid-phase [17,19,21] or liquid-liquid extraction [22,23] to measure BUP and NorBUP; however, these methods are time-consuming although liquid-liquid extraction can be quite efficient and also cost-efficient. Moreover, extraction of glucuronides into a lipophilic organic solvent is difficult due to their highly polar and hydrophilic properties [24]. Whereas sample preparation is not necessarily required for urine [18,21,25–27] and oral fluid [28,29], protein precipitation at least is requested for plasma and blood. Interestingly, BUP and NorBUP were previously quantified using protein precipitation in plasma [20]; however, quantification of BUP-GLUC and NorBUP–GLUC were not included in this study. Additionally, BUP and metabolite concentrations were mainly measured in plasma, whether in humans [17,29,30] or rats [31–33] while whole blood [22,34,35] were rarely used. To the best of our knowledge, no study has developed a method of measurement of BUP and metabolites in rat whole blood. To facilitate the conduction of experimental investigations on the mechanisms of BUP-related toxicity, we aimed to develop an LC–MS/MS method allowing the simultaneous quantification of BUP, NorBUP, NLX and their conjugated metabolites in rat whole blood using an extraction based on protein precipitation. We validated the method according to the current acceptance criteria for bioanalytical method validation. This method was used to study the neurorespiratory effects of buprenorphine and ethanol in combination [36].
2. Material and method 2.1. Chemicals and materials BUP Hydrochloride and NLX-3--GLUC were provided by Lipomed (Arlesheim, Switzerland) and dissolved in methanol to obtain concentrations 1 and 0.1 g/L of, respectively. NorBUP, BUP3-D-GLUC, NorBUP–GLUC, BUP-d4, NorBUP-d4 and NLX-d5, were provided by Cerilliant (Austin, TX, USA) and dissolved in methanol to obtain concentrations of 0.1 g/L. NLX hydrochloride dihydrate was provided by LGC (Molshein, France) and dissolved in methanol to obtain a concentration of 0.810 g/L as anhydrous free base. Only three stable-isotope-labeled analogues, BUP-d4, NorBUPd3 and NLX-d5, were used as internal standard (IS). Deuterated analogues of BUP-GLUC, NorBUP-GLUC and NLX-GLUC were not available. Ammonium formate was purchased from Sigma-Aldrich (St Quentin, France). Methanol and acetonitrile were high performance liquid chromatography (HPLC)-grade and obtained from Carlo Erba Reagents (Val de Reuil, France). Water was deionized using milli-Q ultrapure water system (Millipore Corp., Woburn, MA, USA).
In the rat study, BUP hydrochloride powder was generously provided by Reckitt Benckiser (Evry, France) and diluted in 4% Tween® (Sigma-Aldrich, St Quentin, France) for intravenous administration. NLX hydrochloride dihydrate powder (Sigma-Aldrich, France) was diluted in 4% Tween® and mixed with BUP to obtain BUP/NLX solution at 4:1 ratio in agreement with the marketed Suboxone® formulation. 2.2. Preparation of standard solutions Before each experiment, two sets containing stock solutions of BUP, NorBUP, BUP-GLUC, NorBUP-GLUC, NLX and NLX-GLUC were prepared by dilution in methanol at concentrations of 5000, 500, 50 and 5 ng/mL. One set was used to prepare calibration standards and the other to prepare the validation samples and quality controls. IS stock solution (BUP-d4, NorBUP-d3, NLX-d5) was diluted in methanol to obtain a working solution of 250 ng/mL. All these stock solutions were stored at −20 ◦ C. 2.3. Instrumentation LC-MS/MS analysis was performed with the Agilent 1260 infinity HPLC system (Agilent Technology, Santa Clara, CA, USA) consisting of quaternary pumps, an autosampler and a vacuum degasser. Chromatographic separation was carried out using a RaptorTM biphenyl core-shell column (100 mm × 3 mm, 2.7 m) (Restek, Bellefonte, PA, USA) with the corresponding guard column. The HPLC system was coupled to an API 3200TM triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA) equipped with a turboVTM ion source operating in positive electrospray ionization (ESI+ ). Instrument control and data acquisition were performed by Sciex Analyst® 1.6 software. For qualitative results, compound identification is typically performed by retention time (RT) matching and calculating the ratio of quantifier and qualifier MRM transition for each compound. For quantitative results, only quantifier MRM transition was used. 2.4. Method description 2.4.1. Sample preparation Whole blood samples (calibration, validation, internal quality control and unknown specimens) were spiked with 15 L of IS solution. Samples were submitted to protein precipitation by 140 L of acetonitrile and were centrifuged at 18,213x g (12,700 rpm) for 10 min. One hundred L of the supernatant was transferred to a vial containing 300 L of ultrapure water. Vials were then submitted for instrumental analysis. For fortified samples (calibration, validation, internal quality control), blank whole blood (pool of whole blood from 5 nonexposed rats) was spiked with the corresponding working solution at the appropriate concentrations to a final volume of 15 L. For calibration standards, eleven concentrations were used (1, 2.5, 5, 10, 25, 50, 100, 250, 1,000, 2,500 and 5,000 ng/mL). For validation standards, eleven concentrations were used, each in triplicate (0.75, 1.5, 4, 7.5, 15, 40, 75, 150, 750, 1,500 and 4,000 ng/mL). For routine analysis, three concentrations were used for internal quality control (15, 150 and 1,500 ng/mL). 2.4.2. LC conditions The HPLC column was held at 30 ◦ C. The mobile phases consisted of a mixed solvent system of 0.25 mM aqueous ammonium formate with 0.1% formic acid (solvent A) and methanol (solvent B). Gradient elution was performed at a constant 300-L/min flow rate as follows: 1 min equilibration followed by a linear decrease during 7 min from 95 to 5% of solvent A and 2 min maintained at 5% of solvent A (see Table 1S in the Supplementary material). Then,
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the system was placed in the initial mobile phase conditions for 3.5 min to allow column equilibration. The injection volume was set at 80 L in full loop mode. Autosampler was kept at 8 ◦ C. The system pressure was near 275 bars at the beginning of the run. 2.4.3. MS/MS conditions Detection was carried out using scheduled MRM mode. However, surviving precursor ions were monitored to reduce lack of sensitivity of fragmented ions. Collision energies were optimized to monitor the two most abundant transitions per compound and used for identification and determination. Nitrogen was used as desolvation gas. Capillary voltage was set at 5,500 V and source temperature at 500 ◦ C. Dwell time was 62.5 ms for each MRM transition. 2.5. Method validation For validation, a “fitness-to-purpose” strategy was used based on the accuracy profile procedure [37]. The described procedure was designed as recommended for any new analytical method used in pharmacokinetic studies [38] and validated according to the accepted recommendations [38–42]. Three independent series (different days and different operators) were conducted and analyzed. On each validation day, calibration standards and validation samples, in triplicate (n = 9 for each level), were prepared independently from the corresponding standard solutions. The concentrations of compounds in validation samples were calculated from the area ratio between the compound and its corresponding IS, based on calibration curves prepared daily as described below. BUP-d4 was used as IS for BUP and BUP-GLUC quantification, NorBUP-d3 for NorBUP and NorBUP-GLUC quantification and NLX-d5 for NLX and NLX-GLUC quantification. Data were obtained using the e-noval® software (Arlenda® , Liege, Belgium). The acceptance limits were set at ± 40% for all the studied compounds except for NLX-GLUC (±50%). The -expectation tolerance interval was set at ± 10%, selected according to the intended use of the analytical procedure. 2.5.1. Selectivity and carry over The method selectivity toward matrix was determined by checking the absence of interfering peaks at compound RT in blank whole blood from six different batches. Carry-over effects were evaluated by injecting the mobile phase at initial conditions [buffer/methanol (95:5, v/v)] after the highest calibration sample (5000 ng/mL) and by checking the absence of peak compounds. 2.5.2. Calibration models, LLOQs, ULOQs and LODs Calibration models, limits of detection (LODs), lower limits of quantification (LLOQs) and upper limits of quantification (ULOQs) for each compound were calculated using the e-noval software based on three validation series performed on three independent days. LODs and LLOQs were checked from the concentrations of the two transition qualifier and quantifier compounds giving rise to signal-to-noise (S/N) ratios of 3 for a validation sample near the LOD and 10 for a validation sample near the LLOQ as recommended [39–41,43]. MRM ratios of samples near the LOD and LLOQ were calculated as the ratio of the quantifier and qualifier transition and a 30% generic tolerance permitted according to the SANCO/12571/2013 guidelines [41] when compared to mean MRM ratios of the three calibration standards. 2.5.3. Trueness, precision and accuracy profile Quantitative determination of trueness and precision (repeatability and intermediate precision) were assessed by the three validation series, at eleven concentrations of each studied compound ranging from 0.75 to 4000 ng/ml. Ratios between the mean concentrations obtained from the analytical measurements and
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the theoretical concentrations were used to calculate trueness. Variances of repeatability (intra-day variances) and intermediate precision (sum of intra-day and inter-days variances) were used to estimate the precision of measurements [44]. Precision at each concentration was expressed as the coefficient of variation (CV) based on the theoretical value of the concentration [45]. According to recommendations, trueness was expected to be within 85–115 % of the theoretical value (LLOQ 80–120 %) and precision within ± 15% except for LLOQ (20%). Accuracy profiles of all the compounds were used to determine the total error including systematic (trueness) and random (intermediate precision) errors. 2.5.4. Freeze/thaw stability at −20 ◦ C All the whole blood samples were frozen after blood collection and underwent one freeze/thaw cycle. All the studied compounds were reported to be stable over at least 9 months in whole blood or plasma [23,46–49]. This delay between blood sampling and LC–SM2 analysis was observed in our study. Additionally, according to the literature [38], assessment of compound stability in our study was not requested. 2.5.5. Precipitation recovery and matrix effect As whole blood components are able to disturb quantification of compounds by signal suppression or enhancement, matrix effect was quantitatively evaluated, based on Matuszewski’s approach [50]. Samples at intermediate (150 ng/mL) concentrations were analyzed. IS concentrations were set at fixed concentrations (250 ng/mL) for all samples based on our procedure. Three different sample sets were prepared: (1) water samples spiked after precipitation in triplicate; (2) five different whole blood samples spiked after protein precipitation and (3) before protein precipitation. The matrix effect of each compound was evaluated as the peak area ratio of (2) to (1) and was expressed as value above (enhancement) or below (suppression) 100 %. Precipitation recovery was estimated as the peak area ratio of (3) to (2) and was expressed as value above or below 100 %. For (1), the mean peak area of each triplicate was used as reference. For the five different rat whole blood (2 and 3), the mean peak area was determined. For BUP, NBUP and NLX, the corrected matrix effect was calculated using area ratio of the area quantifier to area of the internal standard. Variability between whole blood was expressed as CV. Variability of ME between five different sources of blank whole blood matrix was expected not to exceed 15% CV, but larger variability was acceptable if stable-isotope-labeled analogues are used as IS that effectively compensate for variability in matrix effect. [51–54]. 2.6. Determination of rat whole blood concentrations of BUP, NLX and their metabolites 24 h after intravenous BUP/NLX administration This experiment was a part of a larger pharmacokinetic study conducted in our laboratory. Our experimental protocol was approved by our animal institutional ethic committee (Paris-Descartes University). We used one 7-week-old male Sprague-Dawley rat weighing 300 g and purchased from Janvier (Genest, France). Twenty-four hours before the drug administration, the rat was anesthetized with intraperitoneal 70 mg.kg−1 ketamine (Ketalar® ) and 10 mg.kg1 xylazine (Rompum® ) and its femoral vein and artery catheterized, as previously reported [5]. BUP/NLX (30.0/7.5 mg/kg) was administered by the intravenous route and one blood sample (150 L) collected 24 h after drug administration by the arterial route. The sample was homogenized and frozen immediately at −20◦ c until LC-MS/MS analysis. Before analysis, the sample was spiked with the IS solutions and treated similarly to the calibration and validation samples.
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Table 1 Multiple Reaction Monitoring (MRM) transitions, cone voltage, collision energy and retention time of the different studied compounds and internal standards (italic data). The quantification transitions are underlined. Internal standards were used for each compounds.
Compounds
Retention time (min)
Buprenorphine 1
7.4
Norbuprenorphine 2 Buprenorphine-3-glucuronide
6.8 1
6.6
Norbuprenorphine-3-glucuronide 2
5.9
Naloxone 3
5.1
Naloxone-3-glucuronide 3
4.4
Buprenorphine-d4 1 Norbuprenorphine-d3 2 Naloxone-d5 3
7.4 6.8 5.1
MRM transitions (m/z)
Cone voltage (eV)
Collision Energy (eV)
468.2 - 396.3 468.2 - 468.2 414.2 - 414.2 414.2 - 83.2 645.1 - 469.4 645.1 - 645.4 590.4 - 590.4 590.4 - 414.4 328.2 - 328.2 328.2 - 310.2 504.2 - 504.2 504.2 - 414.4 472.2 - 400.2 417.2 - 165.0 333.2 - 315.1
91 91 91 91 106 106 101 101 51 51 101 101 91 91 51
47 21 17 65 55 25 23 41 15 23 23 41 47 95 23
The exponent allow linking each compound to its internal standard used for quantification.
3. Results and discussion 3.1. Method development 3.1.1. Sample preparation We used a simple cost- and time-saving extraction technique based on protein precipitation, which additionally allowed optimizing recoveries of glucuronide molecules. We tested two solvents (methanol and acetonitrile) at different dilution ratios. We did not test other protein precipitation techniques in order to preserve interface cleanness and avoid alterations in glucuronide metabolites. Acetonitrile was finally chosen for proteins precipitation, as recommended with acetonitrile-to-plasma ratio of 3:1 (showing high protein precipitation efficiency of 93.5% in this setting) [55]. Furthermore, isotope-labeled IS were used as recommended to improve the method’s selectivity and robustness validation performance [48–51]. Supernatants were reconstituted with water before injection to near the initial phase composition of the chromatographic method and thus to limit the impact of the solvent concentration in the injected specimen on the peak resolution. 3.1.2. LC conditions Due to the heterogeneity of the physicochemical properties of the studied compounds, elution parameters had to be determined to obtain, in the same analysis, sufficient robustness, selectivity, resolution and retention for each analyte. Retention was optimized to compensate for the lower selectivity due to monitoring of the precursor ion and for the sample treatment performed by protein precipitation that gave less purified extracts. A RaptorTM biphenyl core-shell column was chosen because of its ability to provide highly retentive, selective and rugged reversed-phase separations of hydrophilic and lipophilic drugs and metabolites such as BUP [56]. By increasing retention of conjugated compounds, this column could reduce ion suppression. Polar methanol mobile phase was used according to Restek’s recommendations for increasing selectivity of the RaptorTM biphenyl column by pi () stacking. Various parameters were tested including the mobile phase pH (buffer and acid adjunction), the flow rate, the equilibration time and the gradient elution. Since the chosen conditions are a compromise between the impacts of each parameter on the retention of each substance, we have chosen not to detail all implemented tests. The molarity of ammonium formate buffer was tested between 0.25 and 5 mM. The best selectivity and RT stability were obtained at 0.25 mmol/L adjusted to pH 2.7 (which is in accordance with most
published methods used to quantify BUP [17,25,30]). In these conditions, naloxone was sufficiently retained to obtain an acceptable resolution while preserving the retention of the other compounds. In the final chromatographic conditions, analytes were separated in <8 min which was faster than in many published methods using LC–MS/MS for BUP quantification [14,17,19,57]. The retention times of BUP, NLX and their metabolites are listed in Table 1. In these conditions, no interfering peak was observed (See selectivity and carry over). As recommended [51], isotope-labeled IS were used to improve the robustness of the method. 3.1.3. MS/MS conditions For each studied compound, ionization parameters, cone voltages and collision energies were optimized by infusion of a 100 ng/mL solution into the detector at 10 L/min flow rate in combination with the mobile phase. The positive mode was chosen. In a first step, fragmented ions with highest signals were chosen for MRM quantification. However, this detection method resulted in a poor sensitivity that was not in accordance with the expected LLOQ. To compensate this lack of sensibility, surviving parent ions with higher signals than their respective fragmented ions were monitored as previously reported for the detection of BUP [30,35,58]. Despite a decrease in selectivity compared to the usual MRM mode which detects fragment ions, monitoring parent ions improved sensitivity. Optimized qualifier and quantifier MRM transitions, cone voltage and collision energies are shown in Table 1. 3.1.4. Selectivity and carry over No interference was observed at the RT of the studied compounds and IS, based on six different blank whole blood (see Fig. 1S in the Supplementary material). No carry over (0.01%) was observed when a mobile phase was analyzed just after the highest calibration standard (5000 ng/mL) (data not shown). 3.1.5. Calibration model, LLOQs, ULOQs and LODs Different models of regression were performed by e-noval and the most suitable models based on trueness and precision are listed in Table 2. Weighed regression models were more appropriate for these calibration ranges (one or two order of magnitude). The regression equations, LODs, LLOQs and ULOQs are listed in Table 2. LODs and LLOQs were calculated by e-noval and confirmed by analyzing S/N of 3 near LD. S/N near LODs and LLOQs were all superior to 3 and 10, respectively for all the compounds (See Fig. 1S and Table 2S in the Supplementary material) and were in accordance with the international recommendations for validation of analytical methods [39–41,43]. MRM ratios at validation samples near
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Fig. 1. Multiple reaction monitoring (MRM) chromatograms for each compound at a concentration between its limit of detection (LOD) and its lower limit of quantification (LLOQ) (quantifier transitions in blue and qualifier transitions in pink) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
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Table 2 Regression model, range, limit of detection (LOD) and lower limit of quantification (LLOQ) for each studied compound. All ULOQs were near the highest calibration standard (i.e. 4000 ng/mL). All R2 were > 0.998. Compounds
Regression model
Range (ng/mL)
LOD (ng/mL)
LLOQ (ng/mL)
Buprenorphine Norbuprenorphine Buprenorphine-glucuronide Norbuprenorphine-glucuronide Naloxone Naloxone-glucuronide
1/x weighed quadratic 1/x2 weighed quadratic 1/x weighed quadratic 1/x2 weighed quadratic 1/x2 weighed quadratic log linear
6.9-4000 6.2-4000 3.6-4000 3.3-4000 1.3-4000 57.7-4000
1.4 0.5 1.1 0.4 0.4 17.5
6.9 6.2 3.6 3.3 1.3 57.7
LOD, limit of detection; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification.
Table 3 Precision and trueness at each validation level (N = 9 for each concentration). Values in italic represent non-validated data. Concentrations (ng/mL)
Buprenorphine LLOQ = 6.9 ng/mL
Precision (CV (%))
Trueness (%)
Rep IP 0.75 1.5 4.0 7.5 15 40 75 150 750 1500 4000
ND 14.1 5.1 10.3 3.6 4.2 3.5 3.9 1.5 1.7 4.2
ND 60.4 15.1 11.3 4.1 5.9 4.3 7.7 8.9 6.5 5.5
Norbuprenorphine LLOQ = 6.2 ng/mL
Naloxone LLOQ = 1.3 ng/mL
Buprenorphineglucuronide LLOQ = 3.6 ng/mL
Norbuprenorphineglucuronide LLOQ = 3.3 ng/mL
Naloxoneglucuronide LLOQ = 57.7 ng/mL
Precision (CV (%))
Precision (CV (%))
Precision (CV (%))
Precision (CV (%))
Precision (CV (%))
Trueness (%)
Rep IP ND 71.5 85.0 87.7 95.8 96.6 91.6 96.2 96.3 95.6 95.0
15.3 12.7 6.8 7.7 4.9 5.5 2.9 6.1 4.5 3.7 9.8
125.5 61.6 22.4 8.1 7.0 7.5 7.6 6.8 4.8 3.7 9.8
Trueness (%)
Rep IP 30.7 63.0 92.4 98.9 108.7 106.0 104.7 107.1 97.1 91.6 98.2
9.5 12.7 4.9 6.1 4.0 6.6 6.7 7.5 6.4 5.2 8.4
20.5 14.7 9.3 6.9 8.3 9.2 8.8 7.6 6.4 6.2 9.1
Trueness (%)
Rep IP 118.6 99.1 97.4 90.7 97.7 102.5 99.6 103.2 98.5 96.4 100.9
ND 11.8 14.3 9.6 4.2 5.9 6.9 9.8 7.6 2.7 2.8
ND 30.8 14.3 9.6 6.0 5.9 8.2 13.7 10.5 2.9 2.8
Trueness (%)
Rep IP ND 65.3 94.5 92.1 96.7 91.7 93.4 97.0 95.8 99.4 99.1
19.5 33.5 9.9 6.4 6.2 5.3 5.7 7.0 7.4 4.7 3.9
22.8 34.1 14.2 7.8 14.3 8.6 8.4 7.4 7.4 6.0 4.8
Trueness (%)
Rep IP 93.3 119.6 97.3 98.8 101.5 104.5 102.7 106.5 109.3 110.0 102.1
ND ND ND ND ND 19.3 9.0 11.9 11.1 5.3 7.6
ND ND ND ND ND 22.3 9.0 14.9 13.2 12.9 8.4
ND ND ND ND ND 121.6 114.1 105.6 105.5 102.0 115.7
LLOQ, lower limit of quantification; Rep, repeatability; IP, intermediate precision; CV, coefficient of variation; ND, not determined.
Table 4 Precipitation recovery and matrix effects expressed as value above or below 100% (CV%) at intermediate concentrations (150 ng/mL) for each studied compound in rat whole blood and at fixed concentration (250 ng/mL) for the three internal standards.
Buprenorphine Norbuprenorphine Naloxone Buprenorphine-glucuronide Norbuprenorphine-glucuronide Naloxone-glucuronide Buprenorphine-d4 Norbuprenorphine-d3 Naloxone-d5
Precipitation recovery % (% CV) n=5
Matrix effect % (% CV) n=5
Corrected Matrix effect % (% CV) n=5
109 (19) 98 (26) 104 (3) 98 (33) 101 (6) 103 (2) 91 (22) 111 (20) 98 (14)
58 (21) 82 (8) 78 (5) 100 (6) 107 (6) 101 (3) 48 (28) 55 (18) 78 (6)
94 (11) 93 (6) 104 (14) / / / / / /
the LODs and LLOQs were all within the 30% tolerance levels compared to the calibration standards (Fig. 1). Considering low whole blood volumes (50 L), LLOQs and ULOQs of all the compounds of interest were suitable for BUP pharmacokinetic studies in the rat. ULOQs were near the highest calibration standard (i.e. 4000 ng/mL) for each compound.
3.1.7. Precipitation recovery and matrix effects In the rat whole blood, precipitation recovery and matrix effect ranged between 91–109 % and 48–101 %, respectively (Table 4). For BUP, NBUP and NLX, the corresponding IS presented similar results. The ion suppression relative to BUP (evidenced by matrix effect of 58% (CV, 21%) was compensated by the addition of stable isotope-labeled analogues used as IS during the validation experiment (corrected matrix effect of 94% (CV, 11%)). Ion suppression of NBUP and NLX was reduced due to their deuterated analogues.
3.1.6. Trueness, precision and accuracy profiles Trueness and precision are shown in Table 3. Trueness for all the studied compounds was within 85–115 % of the theoretical value (LLOQ 80–120 %). Precision was within ±15% CV (LLOQ 20%) [39–41,43]. Regarding the calibration range, the obtained profiles were within the acceptance limits of ± 40% (50% for NLX-GLUC) proposed for toxicological analysis. The accuracy profiles of each compound are illustrated in Fig. 2S in the Supplementary material.
3.1.8. Determination of rat whole blood concentrations of BUP, NLX and their metabolites 24 h after intravenous BUP/NLX administration The developed LC-MS/MS method was used to investigate concentrations of BUP, NorBUP, NLX and their glucuronide metabolites, 24 h after the intravenous administration of 30/7.5 mg/kg BUP/NLX in a Sprague Dawley rat. The concentrations obtained were the followings: 19.1 ng/ml of BUP, 15.3 ng/ml of NorBUP, 131 ng/ml
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Fig. 2. Chromatographic profile of a rat whole blood sample 24 h after the intravenous administration of 30/7.5 mg/kg buprenorphine/naloxone combination. The obtained concentrations were the following: 19.1 ng/ml of buprenorphine, 15.3 ng/ml of norbuprenorphine, 131 ng/ml of buprenorphine-glucuronide, 102 ng/ml of norbuprenorphineglucuronide, 7.4 ng/ml of naloxone and 25.7 ng/ml of naloxone-glucuronide.
of BUP-GLUC, 102 ng/ml of NorBUP-GLUC, 7.37 ng/ml of NLX and 25.7 ng/ml of NLX-GLUC. The chromatographic profiles of the different compounds are shown in Fig. 2. When considering the large compendium of the French cases of BUP-attributed fatalities [59], blood levels for BUP and norBUP ranged from 0.1 to 76 ng/ml and <0.1 to 65 ng/ml, respectively, supporting that 30 mg/kg BUP dosage in the rat was realistic to obtain concentrations in the range of those observed in humans in the presence of toxicity or fatality. Thus, our assay which successfully achieved to measure BUP, naloxone and their metabolites in the rat blood, seems reliable for BUP toxicity investigation in humans although we cannot rule out a lack of sensitivity for the measurement of blood concentrations from suspected drug users with expected lower values (mean values of 1.7 and 1.0 ng/g, respectively, for BUP and NorBUP [58]).
ing. Olivier Roussel: Conceptualization, Supervision, Methodology, Writing - original draft. Declaration of Competing Interest None. Acknowledgement The authors would like to thank the animal house laboratory of Paris-Descartes University and to acknowledge Mrs Alison Good, Scotland, UK, for her helpful review of the manuscript. Appendix A. Supplementary data
4. Conclusions We developed, fully validated and successfully used a sensitive and accurate LC–MS/MS method of simultaneous quantification of BUP, NLX and their metabolites in rat whole blood with extraction based on protein precipitation. The sample preparation and analytical conditions were optimized to increase sensitivity and limit analysis time and cost. This method is now available to investigate the mechanisms of BUP-related toxicity in rat studies allowing the conduction of complete pharmacokinetic studies. Assessment of our LC–MS/MS method in human whole blood should be of high interest to forensic medicine. Funding This work was supported by the Direction Générale de l’Armement-Mission pour la Recherche et l’Innovation Scientifique (DGA-MRIS) scholarship. CRediT authorship contribution statement Camille Cohier: Investigation, Validation, Formal analysis, Writing - original draft. Sophie Salle: Formal analysis, Writing - original draft. Anne Fontova: Investigation, Validation. Bruno Mégarbane: Conceptualization, Project administration, Writing - review & edit-
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jpba.2019. 113042. References [1] A. Yassen, E. Olofsen, R. Romberg, E. Sarton, L. Teppema, A. Danhof, A. Dahan, Mechanism-based PK/PD modeling of the respiratory depressant effect of buprenorphine and fentanyl in healthy volunteers, Clin Pharmacol. Therap. 81 (2007) 50–58. [2] S.L. Walsh, K.L. Preston, G.E. Bigelow, M.L. Stitzer, Acute administration of buprenorphine in humans: partial agonist and blockade effects, J. Pharmacol. Exp. Ther. 274 (1995) 361–372. [3] D.R. Jasinski, J.S. Pevnick, J.D. Griffith, Human pharmacology and abuse potential of the analgesic buprenorphine: a potential agent for treating narcotic addiction, Arch. Gen. Psychiatry 35 (1978) 501–516. [4] L. Chevillard, B. Mégarbane, P. Risède, F.J. Baud, Characteristics and comparative severity of respiratory response to toxic doses of fentanyl, methadone, morphine, and buprenorphine in rats, Toxicol. Lett. 191 (2009) 327–340. [5] C. Cohier, L. Chevillard, P. Risède, O. Roussel, B. Mégarbane, Respiratory effects of buprenorphine/naloxone alone and in combination with diazepam in naive and tolerant rats, Toxicol. Lett. 228 (2014) 75–84. [6] B. Mégarbane, A. Buisine, F. Jacobs, D. Résière, L. Chevillard, E. Vicaut, F.J. Baud, Prospective comparative assessment of buprenorphine overdose with heroin and methadone: clinical characteristics and response to antidotal treatment, J. Subst. Abuse Treat. 38 (2010) 403–407. [7] O. Ferrant, F. Papin, B. Clin, C. Lacroix, E. Saussereau, J.E. Remoué, J.P. Goullé, Fatal poisoning due to snorting buprenorphine and alcohol consumption, Forensic Sci. Int. 204 (2011) e8–11.
8
C. Cohier, S. Salle, A. Fontova et al. / Journal of Pharmaceutical and Biomedical Analysis 180 (2020) 113042
[8] M. Häkkinen, T. Launiainen, E. Vuori, Benzodiazepines and alcohol are associated with cases of fatal buprenorphine poisoning, Eur. J. Clin. Pharmacol. 68 (2012) 301–309. [9] E.V. Pedapati, S.T. Bateman, Toddlers requiring pediatric intensive care unit admission following at-home exposure to buprenorphine/naloxone, Crit. Care Medicine 12 (2011), e102–107. [10] M. Häkkinen, P. Heikman, I. Ojanperä, Parenteral buprenorphine-naloxone abuse is a major cause of fatal buprenorphine-related poisoning, Forensic Sci. Int. 232 (2013) 11–15. [11] E.R. Garrett, V.R. Chandran, Pharmacokinetics of morphine and its surrogates VI: bioanalysis, solvolysis kinetics, solubility, pK’a values, and protein binding of buprenorphine, J. Pharm. Sci. 74 (1985) 515–524. [12] K. Kobayashi, T. Yamamoto, K. Chiba, M. Tani, N. Shimada, T. Ishizaki, Y. Kuroiwa, Human buprenorphine N-dealkylation is catalyzed by cytochrome P450 3A4, Drug Metab. Dispos. 26 (1998) 818–821. [13] B. Mégarbane, N. Marie, S. Pirnay, S.W. Borron, P.N. Gueye, P. Risède, C. Monier, F. Noble, F.J. Baud, Buprenorphine is protective against the depressive effects of norbuprenorphine on ventilation, Toxicol. Appl. Pharmacol. 212 (2006) 256–267. [14] S.M. Brown, M. Holtzman, T. Kim, Buprenorphine metabolites, buprenorphine-3-glucuronide and norbuprenorphine-3-glucuronide, are biologically active, Anesthesiology 115 (2011) 1251–1260. [15] A. Di Marco, M. D’Antoni, S. Attaccalite, Determination of drug glucuronidation and UDP-glucuronosyltransferase selectivity using a 96-well radiometric assay, Drug Metab. Dispos. 33 (2005) 812–819. [16] T. Seldén, J. Ahlner, H. Druid, R. Kronstrand, Toxicological and pathological findings in a series of buprenorphine related deaths. Possible risk factors for fatal outcome, Forensic Sci. Int. 2220 (2012) 284–290. [17] A. Ceccato, R. Klinkenberg, P. Hubert, Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry, J. Pharm. Biomed. Anal. 32 (2003) 619–631. [18] D.S. Harris, J.E. Mendelson, E.T. Lin, Pharmacokinetics and subjective effects of sublingual buprenorphine, alone or in combination with naloxone: lack of dose proportionality, Clin. Pharmacokinetics 43 (2004) 329–340. [19] M. Concheiro, D.M. Shakleya, M.A. Huestis, Simultaneous analysis of buprenorphine, methadone, cocaine, opiates and nicotine metabolites in sweat by liquid chromatography tandem mass spectrometry, Anal. Bioanal. Chem. 400 (2011) 69–78. [20] G. Lüthi, V. Blangy, C.B. Eap, Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry, J. Pharm. Biomed. Anal. 77 (2013) 1–8. [21] M.J. Swortwood, K.B. Scheidweiler, A.J. Barnes, L.M. Jansson, M. Huestis, Simultenous quantification of buprenorphine, naloxone and phase I and II metabolites in plasma and breastmilk by liquid chromatography-tandem mass spectrometry, J. Chromatogr. A 1446 (2016) 70–77. [22] A. Tracqui, P. Kintz, P. Mangin, HPLC/MS determination of buprenorphine and norbuprenorphine in biological fluids and hair samples, J. Forensic Sci. 42 (1997) 111–114. [23] D.E. Moody, M.H. Slawson, E.C. Strain, J.D. Laycock, A.C. Spanbauer, R.L. Foltz, A liquid chromatographic-electrospray ionization-tandem mass spectrometric method for determination of buprenorphine, its metabolite, norbuprenorphine, and a coformulant, naloxone, that is suitable for in vivo and in vitro metabolism studies, Anal. Biochem. 306 (2002) 31–39. [24] J. Trontelj, Quantification of glucuronides metabolites in biological matrices by LC-MSMS, in: J. Prasain (Ed.), Tandem Mass Spectrometry – Application and Principles, 2012, pp. 531–556, 2012 (accessed 07/08/ 2019) In Tech, Open Access http://www.intechopen.com/books/tandem-mass-spectrometryapplications-and-principles/quantification-of-glucuronide-metabolites-inbiological-matrices-by-lc-ms-ms. [25] S. Hegstad, H.Z. Khiabani, E.L. Øiestad, Rapid quantification of buprenorphine-glucuronide and norbuprenorphine-glucuronide in human urine by LC-MS-MS, J. Anal. Toxicol. 31 (2007) 214–219. [26] S.J. Marin, G.A. McMillin, Quantitation of buprenorphine, Norbuprenorphine, buprenorphine glucuronide, Norbuprenorphine Glucuronide, and naloxone in urine by LC-MS/MS, Methods Mol. Biol. 1383 (2016) 69–78. [27] M. Agostini, C. Renzoni, E. Pierini, M. Piergiovanni, V. Termopoli, G. Famiglini, P. Palma, A. Cappiello, Rapid, hydrolysis-free, dilute-and-shoot method for the determination of buprenorphine, norbuprenorphine and their glucuronides in urine samples using UHPLC-MS/MS, J. Pharm. Biomed. Anal. 166 (2019) 236–243. [28] M.T. Truver, M.J. Swortwood, Quantitative analysis of novel synthetic opioids, morphine and buprenorphine in oral fluid by LC-MS-MS, J. Anal. Toxicol. 42 (2018) 554–561. [29] Y. Liu, X. Li, A. Xu, A.F. Nasser, C. Heidbreder, Simultaneous determination of buprenorphine, norbuprenorphine and naloxone in human plasma by liquid chromatography/tandem mass spectrometry, J. Pharm. Biomed. Anal. 120 (2016) 142–152. [30] A. Polettini, M.A. Huestis, Simultaneous determination of buprenorphine, norbuprenorphine, and buprenorphine-glucuronide in plasma by liquid chromatography-tandem mass spectrometry, J. Chromatogr. B 754 (2001) 447–459. [31] S. Gopal, T.-B. Tzeng, A. Cowan, Characterization of the pharmacokinetics of buprenorphine and norbuprenorphine in rats after intravenous bolus administration of buprenorphine, Eur. J. Pharm. Sci. 15 (2002) 287–293.
[32] S. Pirnay, F. Hervé, S. Bouchonnet, Liquid chromatographic-electrospray ionization mass spectrometric quantitative analysis of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in rat plasma, J. Chromatogr. B 41 (2006) 1135–1145. [33] A. Joshi, B. Parris, Y. Liu, C. Heidbreder, P.M. Gerk, M. Halquist, Quantitative determination of buprenorphine, naloxone and their metabolites in rat plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, Biomed. Chromatogr. (2017). [34] H. Hoja, P. Marquet, B. Verneuil, Determination of buprenorphine and norbuprenorphine in whole blood by liquid chromatography-mass spectrometry, J. Anal. Toxicol. 21 (1997) 160–165. [35] D. Favretto, G. Frison, S. Vogliardi, Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor-buprenorphine in urine, blood and hair samples, Rapid Commun. Mass Spectrom. 20 (2006) 1257–1265. [36] C. Cohier, L. Chevillard, S. Salle, P. Risède, O. Roussel, B. Mégarbane, Editor’s Highlight, Neurorespiratory effects of buprenorphine and ethanol in combination: a mechanistic study of drug-Drug interactions in the rat, Toxicol. Sci. 155 (2017) 389–399. [37] M. Feinberg, Validation of analytical methods based on accuracy profiles, J. Chromatogr. A 1158 (2007) 174–183. [38] F.T. Peters, O.H. Drummer, F. Musshoff, Validation of new methods, Forensic Sci. Int. 165 (2007) 216–224. [39] Food and Drug administration, Guidance for Industry. Q2B Validation of Analytical Procedures: Methodology, 1996 (accessed 08/12/ 2019) http:// www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/ guidances/ucm073384.pdf. [40] Food and Drug administration, Guidance for Industry. Bioanalytical Method Validation, 2001 (accessed 08/12/ 2019) http://www.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107. pdf. [41] Commission, Commission decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, Official Journal of the European Communities 2002, 221, 8-35. http://cemu10.fmv.ulg.ac.be/ostc/2002657EC.pdf, 2002 (accessed 08/12/ 2019). [42] S.M.R. Wille, W. Coucke, T. De Baere, F.T. Peters, Update of standard practices for new method validation in forensic toxicology, Curr. Pharm. Des. 23 (2017) 5442–5454. [43] W. Lindner, I.W. Wainer, Requirements for initial assay validation and publication in J. Chromatography B, J. Chromatogr. B 707 (1998) 1–2. [44] E. Chapuzet, N. Mercier, S. Bervoas-Martin, B. Boulanger, P. Chevalier, P. Chiap, D. Grandjean, P. Hubert, P. Lagorce, M. Lallier, M.C. Laparra, M. Laurentie, J.C. Nivet, Chromatographic dosage methods in biological mediums : a validation strategy. Report of an SFSTP commission, STP Pharma. Pratiques 7 (1997) 169–194. [45] E. Rozet, A. Ceccato, C. Hubert, E. Ziemons, R. Oprean, S. Rudaz, B. Boulanger, P. Hubert, Analysis of recent pharmaceutical regulatory documents on analytical method validation, J. Chromatog. A 1158 (2007) 111–125. [46] D.E. Moody, J.D. Laycock, A.C. Spanbauer, D.J. Crouch, R.L. Foltz, J.L. Josephs, L. Amass, W.K. Bickel, Determination of buprenorphine in human plasma by gas chromatography-positive ion chemical ionization mass spectrometry and liquid chromatography-tandem mass spectrometry, J. Anal. Toxicol. 21 (1997) 406–414. [47] K.A. Hadidi, J.S. Oliver, Stability of morphine and buprenorphine in whole blood, Int. J. Legal Med. 111 (1998) 165–167. [48] H. Jiang, Y. Wang, M.S. Shet, Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma, J. Chromatogr. B 879 (2011) 2663–2668. [49] Y. Liu, X. Li, A. Xu, Simultaneous determination of buprenorphine, norbuprenorphine and naloxone in human plasma by liquid chromatography/tandem mass spectrometry, J. Pharm. Biomed. Anal. 120 (2016) 142–152. [50] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS, Anal. Chem. 75 (2003) 3019–3030. [51] C.T. Viswanathan, S. Bansal, B. Booth, A.J. DeStefano, M.J. Rose, J. Sailstad, V.P. Shah, J.P. Skelly, P.G. Swann, R. Weiner, Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays, Pharm. Res. 10 (2007) 1962–1973. [52] E. Stokvis, H. Rosing, J.H. Beijnen, Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun. Mass Spectrom. 19 (2005) 401–407. [53] T. Berg, D.H. Strand, 1 3 C labelled internal standards–a solution to minimize ion suppression effects in liquid chromatography-tandem mass spectrometry analyses of drugs in biological samples? J. Chromatogr. A 1218 (2011) 9366–9374. [54] S. Wang, M. Cyronak, E. Yang, Does a stable isotopically labeled internal standard always correct analyte response? A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma, J. Pharm. Biomed. Anal. 43 (2007) 701–707. [55] C. Polson, P. Sarkar, B. Incledon, Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry, J. Chromatogr. B 785 (2003) 263–275.
C. Cohier, S. Salle, A. Fontova et al. / Journal of Pharmaceutical and Biomedical Analysis 180 (2020) 113042 ¨ [56] Restek. Colonnes LC c¨ ore-shellRaptor Biphenyl. https://clicktime.symantec. com/3LVnuhm5qWRJziAMCNKxaDf6H2?u=https%3A%2F%2Fwww.restek. fr%2Fcatalog%2Fview%2F40873 (accessed 08/12/ 2019). [57] Y. Chang, D.E. Moody, Effect of benzodiazepines on the metabolism of buprenorphine in human liver microsomes, Eur. J. Clin. Pharmacol. 60 (2005) 875–881.
[58] T. Seldén, M. Roman, H. Druid, LC-MS-MS analysis of buprenorphine and norbuprenorphine in whole blood from suspected drug users, Forensic Sci. Int. 209 (2011) 113–119. [59] P. Kintz, Deaths involving buprenorphine: a compendium of French cases, Forensic Sci. Int. 121 (2001) 65–69.
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Determination of buprenorphine, naloxone and phase I and phase II metabolites in rat whole blood by LC–MS/MS Camille Cohier a,b,c,d , Sophie Salle d , Anne Fontova d , Bruno Mégarbane a,b,c,e,∗ , Olivier Roussel a,b,c,d a
Inserm, U1144, Paris, France Paris-Descartes University, UMR-S 1144, Paris, France c Paris-Diderot University, UMR-S 1144, Paris, France d Forensic Toxicology Unit, Forensic Sciences Institute of the French Gendarmerie, Pontoise, France e Department of Medical and Toxicological Critical Care, Lariboisière Hospital, Paris, France b
a r t i c l e
i n f o
Article history: Received 7 August 2019 Received in revised form 9 December 2019 Accepted 9 December 2019 Available online 16 December 2019 Keywords: Buprenorphine Naloxone Glucuronide Whole blood Protein precipitation LC–MS/MS
a b s t r a c t Buprenorphine and buprenorphine/naloxone combination are maintenance treatments used worldwide. However, since their marketing, despite ceiling respiratory effects, poisonings and fatalities have been attributed to buprenorphine misuse and overdose. Therefore, to better understand the mechanisms of buprenorphine-related toxicity in vivo, experimental investigations have been conducted, mainly in the rat. We developed a liquid chromatographic-tandem mass spectrometric (LC–MS/MS) method with electrospray ionization for the simultaneous quantification of buprenorphine, naloxone and their metabolites (norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide and naloxone glucuronide) in rat whole blood. Compounds were extracted from whole blood by protein precipitation and chromatographically separated using gradient elution of aqueous ammonium formate and methanol in a Raptor Biphenyl core-shell column (100 mm x 3,0 mm x 2,7 m). Following electrospray ionization, quantification was carried out in the multiple reaction monitoring (MRM) mode by the tandem mass spectrometer API 3200 system. The LC-MS/MS method was validated according to the currently accepted criteria for bioanalytical method validation. The method required small sample volumes (50 L) and was sensitive with limits of quantification of 6.9, 6.2, 3.6, 3.3, 1.3 and 57.7 ng/mL for buprenorphine, norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide, naloxone and naloxone glucuronide respectively. The upper limit of quantification was 4000 ng/ml for all the studied compounds. Trueness (88–115 %), repeatability and intermediate precision (both <15%) were in accordance with the international recommendations. The procedure was successfully used to quantify these compounds in the whole blood sample from one rat 24 h after the intravenous administration of buprenorphine/naloxone (30.0/7.5 mg/kg). © 2019 Elsevier B.V. All rights reserved.
1. Introduction Buprenorphine (BUP), a semi-synthetic opioid derivate of thebaine, is widely used as an effective maintenance treatment in heroin addicts. Since 2006, a BUP/naloxone (BUP/NLX) combination has been marketed with the aim of preventing intravenous misuse of crushed buprenorphine tablets. In humans [1–3] as well as rodents [4,5], BUP was shown to exhibit ceiling respiratory effects in contrast to other opioids, supporting its safety profile. However,
∗ Corresponding author at: Department of Medical and Toxicological Critical Care, Lariboisière Hospital, 2 Rue Ambroise Paré 75010, Paris, France. E-mail address: [email protected] (B. Mégarbane). https://doi.org/10.1016/j.jpba.2019.113042 0731-7085/© 2019 Elsevier B.V. All rights reserved.
reports emerged of BUP-associated poisonings with typical opioid features and respiratory depression and even fatalities [6–8] while toxicity prevention using the BUP/NLX combination remains a matter of debate [9,10]. BUP is highly bound to blood proteins (96%) and metabolized in the liver by the cytochrome P450 3A4 into the active and toxic metabolite norbuprenorphine (NorBUP) [11–13]. Both compounds are extensively conjugated by UDP-glucuronyl transferase to form the active metabolites, BUP-glucuronide (BUP-GLUC) and NorBUP-glucuronide (NorBUP-GLUC) [14]. The mu-opioid receptor antagonist NLX is also conjugated to form an active metabolite, NLX-glucuronide (NLX-GLUC) [15]. In autopsy cases where BUP or NorBUP had been detected in blood or urine, differences in blood concentrations of BUP and NBUP
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between BUP-attributed fatalities and cases with other causes of death were not clinically relevant [7,8,10,16], suggesting that BUP overdose cannot explain the patient’s death by itself in the majority of cases. Mechanisms of BUP-related toxicity remain thus still unclear, requiring extensive investigations based on experimental studies, which mainly involve rats. Due to the small blood volumes sampled from rats, quantification of BUP and its metabolites in whole blood is requested to facilitate the experimental procedures and limit the number of animals in pharmacokinetic studies. Quantification in whole blood is challenging due to the complexity and diversity of the components included in such a matrix, additionally requiring a sensitive and specific detection system such as liquid chromatography coupled to tandem mass spectrometry (LC MS/MS). LC–MS/MS is considered as the technique of reference for drug analysis including BUP in forensic toxicology and situations requiring multiple sampling like pharmacokinetic studies [14,17–20]. Various sample preparation techniques were developed using solid-phase [17,19,21] or liquid-liquid extraction [22,23] to measure BUP and NorBUP; however, these methods are time-consuming although liquid-liquid extraction can be quite efficient and also cost-efficient. Moreover, extraction of glucuronides into a lipophilic organic solvent is difficult due to their highly polar and hydrophilic properties [24]. Whereas sample preparation is not necessarily required for urine [18,21,25–27] and oral fluid [28,29], protein precipitation at least is requested for plasma and blood. Interestingly, BUP and NorBUP were previously quantified using protein precipitation in plasma [20]; however, quantification of BUP-GLUC and NorBUP–GLUC were not included in this study. Additionally, BUP and metabolite concentrations were mainly measured in plasma, whether in humans [17,29,30] or rats [31–33] while whole blood [22,34,35] were rarely used. To the best of our knowledge, no study has developed a method of measurement of BUP and metabolites in rat whole blood. To facilitate the conduction of experimental investigations on the mechanisms of BUP-related toxicity, we aimed to develop an LC–MS/MS method allowing the simultaneous quantification of BUP, NorBUP, NLX and their conjugated metabolites in rat whole blood using an extraction based on protein precipitation. We validated the method according to the current acceptance criteria for bioanalytical method validation. This method was used to study the neurorespiratory effects of buprenorphine and ethanol in combination [36].
2. Material and method 2.1. Chemicals and materials BUP Hydrochloride and NLX-3--GLUC were provided by Lipomed (Arlesheim, Switzerland) and dissolved in methanol to obtain concentrations 1 and 0.1 g/L of, respectively. NorBUP, BUP3-D-GLUC, NorBUP–GLUC, BUP-d4, NorBUP-d4 and NLX-d5, were provided by Cerilliant (Austin, TX, USA) and dissolved in methanol to obtain concentrations of 0.1 g/L. NLX hydrochloride dihydrate was provided by LGC (Molshein, France) and dissolved in methanol to obtain a concentration of 0.810 g/L as anhydrous free base. Only three stable-isotope-labeled analogues, BUP-d4, NorBUPd3 and NLX-d5, were used as internal standard (IS). Deuterated analogues of BUP-GLUC, NorBUP-GLUC and NLX-GLUC were not available. Ammonium formate was purchased from Sigma-Aldrich (St Quentin, France). Methanol and acetonitrile were high performance liquid chromatography (HPLC)-grade and obtained from Carlo Erba Reagents (Val de Reuil, France). Water was deionized using milli-Q ultrapure water system (Millipore Corp., Woburn, MA, USA).
In the rat study, BUP hydrochloride powder was generously provided by Reckitt Benckiser (Evry, France) and diluted in 4% Tween® (Sigma-Aldrich, St Quentin, France) for intravenous administration. NLX hydrochloride dihydrate powder (Sigma-Aldrich, France) was diluted in 4% Tween® and mixed with BUP to obtain BUP/NLX solution at 4:1 ratio in agreement with the marketed Suboxone® formulation. 2.2. Preparation of standard solutions Before each experiment, two sets containing stock solutions of BUP, NorBUP, BUP-GLUC, NorBUP-GLUC, NLX and NLX-GLUC were prepared by dilution in methanol at concentrations of 5000, 500, 50 and 5 ng/mL. One set was used to prepare calibration standards and the other to prepare the validation samples and quality controls. IS stock solution (BUP-d4, NorBUP-d3, NLX-d5) was diluted in methanol to obtain a working solution of 250 ng/mL. All these stock solutions were stored at −20 ◦ C. 2.3. Instrumentation LC-MS/MS analysis was performed with the Agilent 1260 infinity HPLC system (Agilent Technology, Santa Clara, CA, USA) consisting of quaternary pumps, an autosampler and a vacuum degasser. Chromatographic separation was carried out using a RaptorTM biphenyl core-shell column (100 mm × 3 mm, 2.7 m) (Restek, Bellefonte, PA, USA) with the corresponding guard column. The HPLC system was coupled to an API 3200TM triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA) equipped with a turboVTM ion source operating in positive electrospray ionization (ESI+ ). Instrument control and data acquisition were performed by Sciex Analyst® 1.6 software. For qualitative results, compound identification is typically performed by retention time (RT) matching and calculating the ratio of quantifier and qualifier MRM transition for each compound. For quantitative results, only quantifier MRM transition was used. 2.4. Method description 2.4.1. Sample preparation Whole blood samples (calibration, validation, internal quality control and unknown specimens) were spiked with 15 L of IS solution. Samples were submitted to protein precipitation by 140 L of acetonitrile and were centrifuged at 18,213x g (12,700 rpm) for 10 min. One hundred L of the supernatant was transferred to a vial containing 300 L of ultrapure water. Vials were then submitted for instrumental analysis. For fortified samples (calibration, validation, internal quality control), blank whole blood (pool of whole blood from 5 nonexposed rats) was spiked with the corresponding working solution at the appropriate concentrations to a final volume of 15 L. For calibration standards, eleven concentrations were used (1, 2.5, 5, 10, 25, 50, 100, 250, 1,000, 2,500 and 5,000 ng/mL). For validation standards, eleven concentrations were used, each in triplicate (0.75, 1.5, 4, 7.5, 15, 40, 75, 150, 750, 1,500 and 4,000 ng/mL). For routine analysis, three concentrations were used for internal quality control (15, 150 and 1,500 ng/mL). 2.4.2. LC conditions The HPLC column was held at 30 ◦ C. The mobile phases consisted of a mixed solvent system of 0.25 mM aqueous ammonium formate with 0.1% formic acid (solvent A) and methanol (solvent B). Gradient elution was performed at a constant 300-L/min flow rate as follows: 1 min equilibration followed by a linear decrease during 7 min from 95 to 5% of solvent A and 2 min maintained at 5% of solvent A (see Table 1S in the Supplementary material). Then,
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the system was placed in the initial mobile phase conditions for 3.5 min to allow column equilibration. The injection volume was set at 80 L in full loop mode. Autosampler was kept at 8 ◦ C. The system pressure was near 275 bars at the beginning of the run. 2.4.3. MS/MS conditions Detection was carried out using scheduled MRM mode. However, surviving precursor ions were monitored to reduce lack of sensitivity of fragmented ions. Collision energies were optimized to monitor the two most abundant transitions per compound and used for identification and determination. Nitrogen was used as desolvation gas. Capillary voltage was set at 5,500 V and source temperature at 500 ◦ C. Dwell time was 62.5 ms for each MRM transition. 2.5. Method validation For validation, a “fitness-to-purpose” strategy was used based on the accuracy profile procedure [37]. The described procedure was designed as recommended for any new analytical method used in pharmacokinetic studies [38] and validated according to the accepted recommendations [38–42]. Three independent series (different days and different operators) were conducted and analyzed. On each validation day, calibration standards and validation samples, in triplicate (n = 9 for each level), were prepared independently from the corresponding standard solutions. The concentrations of compounds in validation samples were calculated from the area ratio between the compound and its corresponding IS, based on calibration curves prepared daily as described below. BUP-d4 was used as IS for BUP and BUP-GLUC quantification, NorBUP-d3 for NorBUP and NorBUP-GLUC quantification and NLX-d5 for NLX and NLX-GLUC quantification. Data were obtained using the e-noval® software (Arlenda® , Liege, Belgium). The acceptance limits were set at ± 40% for all the studied compounds except for NLX-GLUC (±50%). The -expectation tolerance interval was set at ± 10%, selected according to the intended use of the analytical procedure. 2.5.1. Selectivity and carry over The method selectivity toward matrix was determined by checking the absence of interfering peaks at compound RT in blank whole blood from six different batches. Carry-over effects were evaluated by injecting the mobile phase at initial conditions [buffer/methanol (95:5, v/v)] after the highest calibration sample (5000 ng/mL) and by checking the absence of peak compounds. 2.5.2. Calibration models, LLOQs, ULOQs and LODs Calibration models, limits of detection (LODs), lower limits of quantification (LLOQs) and upper limits of quantification (ULOQs) for each compound were calculated using the e-noval software based on three validation series performed on three independent days. LODs and LLOQs were checked from the concentrations of the two transition qualifier and quantifier compounds giving rise to signal-to-noise (S/N) ratios of 3 for a validation sample near the LOD and 10 for a validation sample near the LLOQ as recommended [39–41,43]. MRM ratios of samples near the LOD and LLOQ were calculated as the ratio of the quantifier and qualifier transition and a 30% generic tolerance permitted according to the SANCO/12571/2013 guidelines [41] when compared to mean MRM ratios of the three calibration standards. 2.5.3. Trueness, precision and accuracy profile Quantitative determination of trueness and precision (repeatability and intermediate precision) were assessed by the three validation series, at eleven concentrations of each studied compound ranging from 0.75 to 4000 ng/ml. Ratios between the mean concentrations obtained from the analytical measurements and
3
the theoretical concentrations were used to calculate trueness. Variances of repeatability (intra-day variances) and intermediate precision (sum of intra-day and inter-days variances) were used to estimate the precision of measurements [44]. Precision at each concentration was expressed as the coefficient of variation (CV) based on the theoretical value of the concentration [45]. According to recommendations, trueness was expected to be within 85–115 % of the theoretical value (LLOQ 80–120 %) and precision within ± 15% except for LLOQ (20%). Accuracy profiles of all the compounds were used to determine the total error including systematic (trueness) and random (intermediate precision) errors. 2.5.4. Freeze/thaw stability at −20 ◦ C All the whole blood samples were frozen after blood collection and underwent one freeze/thaw cycle. All the studied compounds were reported to be stable over at least 9 months in whole blood or plasma [23,46–49]. This delay between blood sampling and LC–SM2 analysis was observed in our study. Additionally, according to the literature [38], assessment of compound stability in our study was not requested. 2.5.5. Precipitation recovery and matrix effect As whole blood components are able to disturb quantification of compounds by signal suppression or enhancement, matrix effect was quantitatively evaluated, based on Matuszewski’s approach [50]. Samples at intermediate (150 ng/mL) concentrations were analyzed. IS concentrations were set at fixed concentrations (250 ng/mL) for all samples based on our procedure. Three different sample sets were prepared: (1) water samples spiked after precipitation in triplicate; (2) five different whole blood samples spiked after protein precipitation and (3) before protein precipitation. The matrix effect of each compound was evaluated as the peak area ratio of (2) to (1) and was expressed as value above (enhancement) or below (suppression) 100 %. Precipitation recovery was estimated as the peak area ratio of (3) to (2) and was expressed as value above or below 100 %. For (1), the mean peak area of each triplicate was used as reference. For the five different rat whole blood (2 and 3), the mean peak area was determined. For BUP, NBUP and NLX, the corrected matrix effect was calculated using area ratio of the area quantifier to area of the internal standard. Variability between whole blood was expressed as CV. Variability of ME between five different sources of blank whole blood matrix was expected not to exceed 15% CV, but larger variability was acceptable if stable-isotope-labeled analogues are used as IS that effectively compensate for variability in matrix effect. [51–54]. 2.6. Determination of rat whole blood concentrations of BUP, NLX and their metabolites 24 h after intravenous BUP/NLX administration This experiment was a part of a larger pharmacokinetic study conducted in our laboratory. Our experimental protocol was approved by our animal institutional ethic committee (Paris-Descartes University). We used one 7-week-old male Sprague-Dawley rat weighing 300 g and purchased from Janvier (Genest, France). Twenty-four hours before the drug administration, the rat was anesthetized with intraperitoneal 70 mg.kg−1 ketamine (Ketalar® ) and 10 mg.kg1 xylazine (Rompum® ) and its femoral vein and artery catheterized, as previously reported [5]. BUP/NLX (30.0/7.5 mg/kg) was administered by the intravenous route and one blood sample (150 L) collected 24 h after drug administration by the arterial route. The sample was homogenized and frozen immediately at −20◦ c until LC-MS/MS analysis. Before analysis, the sample was spiked with the IS solutions and treated similarly to the calibration and validation samples.
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Table 1 Multiple Reaction Monitoring (MRM) transitions, cone voltage, collision energy and retention time of the different studied compounds and internal standards (italic data). The quantification transitions are underlined. Internal standards were used for each compounds.
Compounds
Retention time (min)
Buprenorphine 1
7.4
Norbuprenorphine 2 Buprenorphine-3-glucuronide
6.8 1
6.6
Norbuprenorphine-3-glucuronide 2
5.9
Naloxone 3
5.1
Naloxone-3-glucuronide 3
4.4
Buprenorphine-d4 1 Norbuprenorphine-d3 2 Naloxone-d5 3
7.4 6.8 5.1
MRM transitions (m/z)
Cone voltage (eV)
Collision Energy (eV)
468.2 - 396.3 468.2 - 468.2 414.2 - 414.2 414.2 - 83.2 645.1 - 469.4 645.1 - 645.4 590.4 - 590.4 590.4 - 414.4 328.2 - 328.2 328.2 - 310.2 504.2 - 504.2 504.2 - 414.4 472.2 - 400.2 417.2 - 165.0 333.2 - 315.1
91 91 91 91 106 106 101 101 51 51 101 101 91 91 51
47 21 17 65 55 25 23 41 15 23 23 41 47 95 23
The exponent allow linking each compound to its internal standard used for quantification.
3. Results and discussion 3.1. Method development 3.1.1. Sample preparation We used a simple cost- and time-saving extraction technique based on protein precipitation, which additionally allowed optimizing recoveries of glucuronide molecules. We tested two solvents (methanol and acetonitrile) at different dilution ratios. We did not test other protein precipitation techniques in order to preserve interface cleanness and avoid alterations in glucuronide metabolites. Acetonitrile was finally chosen for proteins precipitation, as recommended with acetonitrile-to-plasma ratio of 3:1 (showing high protein precipitation efficiency of 93.5% in this setting) [55]. Furthermore, isotope-labeled IS were used as recommended to improve the method’s selectivity and robustness validation performance [48–51]. Supernatants were reconstituted with water before injection to near the initial phase composition of the chromatographic method and thus to limit the impact of the solvent concentration in the injected specimen on the peak resolution. 3.1.2. LC conditions Due to the heterogeneity of the physicochemical properties of the studied compounds, elution parameters had to be determined to obtain, in the same analysis, sufficient robustness, selectivity, resolution and retention for each analyte. Retention was optimized to compensate for the lower selectivity due to monitoring of the precursor ion and for the sample treatment performed by protein precipitation that gave less purified extracts. A RaptorTM biphenyl core-shell column was chosen because of its ability to provide highly retentive, selective and rugged reversed-phase separations of hydrophilic and lipophilic drugs and metabolites such as BUP [56]. By increasing retention of conjugated compounds, this column could reduce ion suppression. Polar methanol mobile phase was used according to Restek’s recommendations for increasing selectivity of the RaptorTM biphenyl column by pi () stacking. Various parameters were tested including the mobile phase pH (buffer and acid adjunction), the flow rate, the equilibration time and the gradient elution. Since the chosen conditions are a compromise between the impacts of each parameter on the retention of each substance, we have chosen not to detail all implemented tests. The molarity of ammonium formate buffer was tested between 0.25 and 5 mM. The best selectivity and RT stability were obtained at 0.25 mmol/L adjusted to pH 2.7 (which is in accordance with most
published methods used to quantify BUP [17,25,30]). In these conditions, naloxone was sufficiently retained to obtain an acceptable resolution while preserving the retention of the other compounds. In the final chromatographic conditions, analytes were separated in <8 min which was faster than in many published methods using LC–MS/MS for BUP quantification [14,17,19,57]. The retention times of BUP, NLX and their metabolites are listed in Table 1. In these conditions, no interfering peak was observed (See selectivity and carry over). As recommended [51], isotope-labeled IS were used to improve the robustness of the method. 3.1.3. MS/MS conditions For each studied compound, ionization parameters, cone voltages and collision energies were optimized by infusion of a 100 ng/mL solution into the detector at 10 L/min flow rate in combination with the mobile phase. The positive mode was chosen. In a first step, fragmented ions with highest signals were chosen for MRM quantification. However, this detection method resulted in a poor sensitivity that was not in accordance with the expected LLOQ. To compensate this lack of sensibility, surviving parent ions with higher signals than their respective fragmented ions were monitored as previously reported for the detection of BUP [30,35,58]. Despite a decrease in selectivity compared to the usual MRM mode which detects fragment ions, monitoring parent ions improved sensitivity. Optimized qualifier and quantifier MRM transitions, cone voltage and collision energies are shown in Table 1. 3.1.4. Selectivity and carry over No interference was observed at the RT of the studied compounds and IS, based on six different blank whole blood (see Fig. 1S in the Supplementary material). No carry over (0.01%) was observed when a mobile phase was analyzed just after the highest calibration standard (5000 ng/mL) (data not shown). 3.1.5. Calibration model, LLOQs, ULOQs and LODs Different models of regression were performed by e-noval and the most suitable models based on trueness and precision are listed in Table 2. Weighed regression models were more appropriate for these calibration ranges (one or two order of magnitude). The regression equations, LODs, LLOQs and ULOQs are listed in Table 2. LODs and LLOQs were calculated by e-noval and confirmed by analyzing S/N of 3 near LD. S/N near LODs and LLOQs were all superior to 3 and 10, respectively for all the compounds (See Fig. 1S and Table 2S in the Supplementary material) and were in accordance with the international recommendations for validation of analytical methods [39–41,43]. MRM ratios at validation samples near
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Fig. 1. Multiple reaction monitoring (MRM) chromatograms for each compound at a concentration between its limit of detection (LOD) and its lower limit of quantification (LLOQ) (quantifier transitions in blue and qualifier transitions in pink) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
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Table 2 Regression model, range, limit of detection (LOD) and lower limit of quantification (LLOQ) for each studied compound. All ULOQs were near the highest calibration standard (i.e. 4000 ng/mL). All R2 were > 0.998. Compounds
Regression model
Range (ng/mL)
LOD (ng/mL)
LLOQ (ng/mL)
Buprenorphine Norbuprenorphine Buprenorphine-glucuronide Norbuprenorphine-glucuronide Naloxone Naloxone-glucuronide
1/x weighed quadratic 1/x2 weighed quadratic 1/x weighed quadratic 1/x2 weighed quadratic 1/x2 weighed quadratic log linear
6.9-4000 6.2-4000 3.6-4000 3.3-4000 1.3-4000 57.7-4000
1.4 0.5 1.1 0.4 0.4 17.5
6.9 6.2 3.6 3.3 1.3 57.7
LOD, limit of detection; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification.
Table 3 Precision and trueness at each validation level (N = 9 for each concentration). Values in italic represent non-validated data. Concentrations (ng/mL)
Buprenorphine LLOQ = 6.9 ng/mL
Precision (CV (%))
Trueness (%)
Rep IP 0.75 1.5 4.0 7.5 15 40 75 150 750 1500 4000
ND 14.1 5.1 10.3 3.6 4.2 3.5 3.9 1.5 1.7 4.2
ND 60.4 15.1 11.3 4.1 5.9 4.3 7.7 8.9 6.5 5.5
Norbuprenorphine LLOQ = 6.2 ng/mL
Naloxone LLOQ = 1.3 ng/mL
Buprenorphineglucuronide LLOQ = 3.6 ng/mL
Norbuprenorphineglucuronide LLOQ = 3.3 ng/mL
Naloxoneglucuronide LLOQ = 57.7 ng/mL
Precision (CV (%))
Precision (CV (%))
Precision (CV (%))
Precision (CV (%))
Precision (CV (%))
Trueness (%)
Rep IP ND 71.5 85.0 87.7 95.8 96.6 91.6 96.2 96.3 95.6 95.0
15.3 12.7 6.8 7.7 4.9 5.5 2.9 6.1 4.5 3.7 9.8
125.5 61.6 22.4 8.1 7.0 7.5 7.6 6.8 4.8 3.7 9.8
Trueness (%)
Rep IP 30.7 63.0 92.4 98.9 108.7 106.0 104.7 107.1 97.1 91.6 98.2
9.5 12.7 4.9 6.1 4.0 6.6 6.7 7.5 6.4 5.2 8.4
20.5 14.7 9.3 6.9 8.3 9.2 8.8 7.6 6.4 6.2 9.1
Trueness (%)
Rep IP 118.6 99.1 97.4 90.7 97.7 102.5 99.6 103.2 98.5 96.4 100.9
ND 11.8 14.3 9.6 4.2 5.9 6.9 9.8 7.6 2.7 2.8
ND 30.8 14.3 9.6 6.0 5.9 8.2 13.7 10.5 2.9 2.8
Trueness (%)
Rep IP ND 65.3 94.5 92.1 96.7 91.7 93.4 97.0 95.8 99.4 99.1
19.5 33.5 9.9 6.4 6.2 5.3 5.7 7.0 7.4 4.7 3.9
22.8 34.1 14.2 7.8 14.3 8.6 8.4 7.4 7.4 6.0 4.8
Trueness (%)
Rep IP 93.3 119.6 97.3 98.8 101.5 104.5 102.7 106.5 109.3 110.0 102.1
ND ND ND ND ND 19.3 9.0 11.9 11.1 5.3 7.6
ND ND ND ND ND 22.3 9.0 14.9 13.2 12.9 8.4
ND ND ND ND ND 121.6 114.1 105.6 105.5 102.0 115.7
LLOQ, lower limit of quantification; Rep, repeatability; IP, intermediate precision; CV, coefficient of variation; ND, not determined.
Table 4 Precipitation recovery and matrix effects expressed as value above or below 100% (CV%) at intermediate concentrations (150 ng/mL) for each studied compound in rat whole blood and at fixed concentration (250 ng/mL) for the three internal standards.
Buprenorphine Norbuprenorphine Naloxone Buprenorphine-glucuronide Norbuprenorphine-glucuronide Naloxone-glucuronide Buprenorphine-d4 Norbuprenorphine-d3 Naloxone-d5
Precipitation recovery % (% CV) n=5
Matrix effect % (% CV) n=5
Corrected Matrix effect % (% CV) n=5
109 (19) 98 (26) 104 (3) 98 (33) 101 (6) 103 (2) 91 (22) 111 (20) 98 (14)
58 (21) 82 (8) 78 (5) 100 (6) 107 (6) 101 (3) 48 (28) 55 (18) 78 (6)
94 (11) 93 (6) 104 (14) / / / / / /
the LODs and LLOQs were all within the 30% tolerance levels compared to the calibration standards (Fig. 1). Considering low whole blood volumes (50 L), LLOQs and ULOQs of all the compounds of interest were suitable for BUP pharmacokinetic studies in the rat. ULOQs were near the highest calibration standard (i.e. 4000 ng/mL) for each compound.
3.1.7. Precipitation recovery and matrix effects In the rat whole blood, precipitation recovery and matrix effect ranged between 91–109 % and 48–101 %, respectively (Table 4). For BUP, NBUP and NLX, the corresponding IS presented similar results. The ion suppression relative to BUP (evidenced by matrix effect of 58% (CV, 21%) was compensated by the addition of stable isotope-labeled analogues used as IS during the validation experiment (corrected matrix effect of 94% (CV, 11%)). Ion suppression of NBUP and NLX was reduced due to their deuterated analogues.
3.1.6. Trueness, precision and accuracy profiles Trueness and precision are shown in Table 3. Trueness for all the studied compounds was within 85–115 % of the theoretical value (LLOQ 80–120 %). Precision was within ±15% CV (LLOQ 20%) [39–41,43]. Regarding the calibration range, the obtained profiles were within the acceptance limits of ± 40% (50% for NLX-GLUC) proposed for toxicological analysis. The accuracy profiles of each compound are illustrated in Fig. 2S in the Supplementary material.
3.1.8. Determination of rat whole blood concentrations of BUP, NLX and their metabolites 24 h after intravenous BUP/NLX administration The developed LC-MS/MS method was used to investigate concentrations of BUP, NorBUP, NLX and their glucuronide metabolites, 24 h after the intravenous administration of 30/7.5 mg/kg BUP/NLX in a Sprague Dawley rat. The concentrations obtained were the followings: 19.1 ng/ml of BUP, 15.3 ng/ml of NorBUP, 131 ng/ml
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Fig. 2. Chromatographic profile of a rat whole blood sample 24 h after the intravenous administration of 30/7.5 mg/kg buprenorphine/naloxone combination. The obtained concentrations were the following: 19.1 ng/ml of buprenorphine, 15.3 ng/ml of norbuprenorphine, 131 ng/ml of buprenorphine-glucuronide, 102 ng/ml of norbuprenorphineglucuronide, 7.4 ng/ml of naloxone and 25.7 ng/ml of naloxone-glucuronide.
of BUP-GLUC, 102 ng/ml of NorBUP-GLUC, 7.37 ng/ml of NLX and 25.7 ng/ml of NLX-GLUC. The chromatographic profiles of the different compounds are shown in Fig. 2. When considering the large compendium of the French cases of BUP-attributed fatalities [59], blood levels for BUP and norBUP ranged from 0.1 to 76 ng/ml and <0.1 to 65 ng/ml, respectively, supporting that 30 mg/kg BUP dosage in the rat was realistic to obtain concentrations in the range of those observed in humans in the presence of toxicity or fatality. Thus, our assay which successfully achieved to measure BUP, naloxone and their metabolites in the rat blood, seems reliable for BUP toxicity investigation in humans although we cannot rule out a lack of sensitivity for the measurement of blood concentrations from suspected drug users with expected lower values (mean values of 1.7 and 1.0 ng/g, respectively, for BUP and NorBUP [58]).
ing. Olivier Roussel: Conceptualization, Supervision, Methodology, Writing - original draft. Declaration of Competing Interest None. Acknowledgement The authors would like to thank the animal house laboratory of Paris-Descartes University and to acknowledge Mrs Alison Good, Scotland, UK, for her helpful review of the manuscript. Appendix A. Supplementary data
4. Conclusions We developed, fully validated and successfully used a sensitive and accurate LC–MS/MS method of simultaneous quantification of BUP, NLX and their metabolites in rat whole blood with extraction based on protein precipitation. The sample preparation and analytical conditions were optimized to increase sensitivity and limit analysis time and cost. This method is now available to investigate the mechanisms of BUP-related toxicity in rat studies allowing the conduction of complete pharmacokinetic studies. Assessment of our LC–MS/MS method in human whole blood should be of high interest to forensic medicine. Funding This work was supported by the Direction Générale de l’Armement-Mission pour la Recherche et l’Innovation Scientifique (DGA-MRIS) scholarship. CRediT authorship contribution statement Camille Cohier: Investigation, Validation, Formal analysis, Writing - original draft. Sophie Salle: Formal analysis, Writing - original draft. Anne Fontova: Investigation, Validation. Bruno Mégarbane: Conceptualization, Project administration, Writing - review & edit-
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jpba.2019. 113042. References [1] A. Yassen, E. Olofsen, R. Romberg, E. Sarton, L. Teppema, A. Danhof, A. Dahan, Mechanism-based PK/PD modeling of the respiratory depressant effect of buprenorphine and fentanyl in healthy volunteers, Clin Pharmacol. Therap. 81 (2007) 50–58. [2] S.L. Walsh, K.L. Preston, G.E. Bigelow, M.L. Stitzer, Acute administration of buprenorphine in humans: partial agonist and blockade effects, J. Pharmacol. Exp. Ther. 274 (1995) 361–372. [3] D.R. Jasinski, J.S. Pevnick, J.D. Griffith, Human pharmacology and abuse potential of the analgesic buprenorphine: a potential agent for treating narcotic addiction, Arch. Gen. Psychiatry 35 (1978) 501–516. [4] L. Chevillard, B. Mégarbane, P. Risède, F.J. Baud, Characteristics and comparative severity of respiratory response to toxic doses of fentanyl, methadone, morphine, and buprenorphine in rats, Toxicol. Lett. 191 (2009) 327–340. [5] C. Cohier, L. Chevillard, P. Risède, O. Roussel, B. Mégarbane, Respiratory effects of buprenorphine/naloxone alone and in combination with diazepam in naive and tolerant rats, Toxicol. Lett. 228 (2014) 75–84. [6] B. Mégarbane, A. Buisine, F. Jacobs, D. Résière, L. Chevillard, E. Vicaut, F.J. Baud, Prospective comparative assessment of buprenorphine overdose with heroin and methadone: clinical characteristics and response to antidotal treatment, J. Subst. Abuse Treat. 38 (2010) 403–407. [7] O. Ferrant, F. Papin, B. Clin, C. Lacroix, E. Saussereau, J.E. Remoué, J.P. Goullé, Fatal poisoning due to snorting buprenorphine and alcohol consumption, Forensic Sci. Int. 204 (2011) e8–11.
8
C. Cohier, S. Salle, A. Fontova et al. / Journal of Pharmaceutical and Biomedical Analysis 180 (2020) 113042
[8] M. Häkkinen, T. Launiainen, E. Vuori, Benzodiazepines and alcohol are associated with cases of fatal buprenorphine poisoning, Eur. J. Clin. Pharmacol. 68 (2012) 301–309. [9] E.V. Pedapati, S.T. Bateman, Toddlers requiring pediatric intensive care unit admission following at-home exposure to buprenorphine/naloxone, Crit. Care Medicine 12 (2011), e102–107. [10] M. Häkkinen, P. Heikman, I. Ojanperä, Parenteral buprenorphine-naloxone abuse is a major cause of fatal buprenorphine-related poisoning, Forensic Sci. Int. 232 (2013) 11–15. [11] E.R. Garrett, V.R. Chandran, Pharmacokinetics of morphine and its surrogates VI: bioanalysis, solvolysis kinetics, solubility, pK’a values, and protein binding of buprenorphine, J. Pharm. Sci. 74 (1985) 515–524. [12] K. Kobayashi, T. Yamamoto, K. Chiba, M. Tani, N. Shimada, T. Ishizaki, Y. Kuroiwa, Human buprenorphine N-dealkylation is catalyzed by cytochrome P450 3A4, Drug Metab. Dispos. 26 (1998) 818–821. [13] B. Mégarbane, N. Marie, S. Pirnay, S.W. Borron, P.N. Gueye, P. Risède, C. Monier, F. Noble, F.J. Baud, Buprenorphine is protective against the depressive effects of norbuprenorphine on ventilation, Toxicol. Appl. Pharmacol. 212 (2006) 256–267. [14] S.M. Brown, M. Holtzman, T. Kim, Buprenorphine metabolites, buprenorphine-3-glucuronide and norbuprenorphine-3-glucuronide, are biologically active, Anesthesiology 115 (2011) 1251–1260. [15] A. Di Marco, M. D’Antoni, S. Attaccalite, Determination of drug glucuronidation and UDP-glucuronosyltransferase selectivity using a 96-well radiometric assay, Drug Metab. Dispos. 33 (2005) 812–819. [16] T. Seldén, J. Ahlner, H. Druid, R. Kronstrand, Toxicological and pathological findings in a series of buprenorphine related deaths. Possible risk factors for fatal outcome, Forensic Sci. Int. 2220 (2012) 284–290. [17] A. Ceccato, R. Klinkenberg, P. Hubert, Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry, J. Pharm. Biomed. Anal. 32 (2003) 619–631. [18] D.S. Harris, J.E. Mendelson, E.T. Lin, Pharmacokinetics and subjective effects of sublingual buprenorphine, alone or in combination with naloxone: lack of dose proportionality, Clin. Pharmacokinetics 43 (2004) 329–340. [19] M. Concheiro, D.M. Shakleya, M.A. Huestis, Simultaneous analysis of buprenorphine, methadone, cocaine, opiates and nicotine metabolites in sweat by liquid chromatography tandem mass spectrometry, Anal. Bioanal. Chem. 400 (2011) 69–78. [20] G. Lüthi, V. Blangy, C.B. Eap, Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry, J. Pharm. Biomed. Anal. 77 (2013) 1–8. [21] M.J. Swortwood, K.B. Scheidweiler, A.J. Barnes, L.M. Jansson, M. Huestis, Simultenous quantification of buprenorphine, naloxone and phase I and II metabolites in plasma and breastmilk by liquid chromatography-tandem mass spectrometry, J. Chromatogr. A 1446 (2016) 70–77. [22] A. Tracqui, P. Kintz, P. Mangin, HPLC/MS determination of buprenorphine and norbuprenorphine in biological fluids and hair samples, J. Forensic Sci. 42 (1997) 111–114. [23] D.E. Moody, M.H. Slawson, E.C. Strain, J.D. Laycock, A.C. Spanbauer, R.L. Foltz, A liquid chromatographic-electrospray ionization-tandem mass spectrometric method for determination of buprenorphine, its metabolite, norbuprenorphine, and a coformulant, naloxone, that is suitable for in vivo and in vitro metabolism studies, Anal. Biochem. 306 (2002) 31–39. [24] J. Trontelj, Quantification of glucuronides metabolites in biological matrices by LC-MSMS, in: J. Prasain (Ed.), Tandem Mass Spectrometry – Application and Principles, 2012, pp. 531–556, 2012 (accessed 07/08/ 2019) In Tech, Open Access http://www.intechopen.com/books/tandem-mass-spectrometryapplications-and-principles/quantification-of-glucuronide-metabolites-inbiological-matrices-by-lc-ms-ms. [25] S. Hegstad, H.Z. Khiabani, E.L. Øiestad, Rapid quantification of buprenorphine-glucuronide and norbuprenorphine-glucuronide in human urine by LC-MS-MS, J. Anal. Toxicol. 31 (2007) 214–219. [26] S.J. Marin, G.A. McMillin, Quantitation of buprenorphine, Norbuprenorphine, buprenorphine glucuronide, Norbuprenorphine Glucuronide, and naloxone in urine by LC-MS/MS, Methods Mol. Biol. 1383 (2016) 69–78. [27] M. Agostini, C. Renzoni, E. Pierini, M. Piergiovanni, V. Termopoli, G. Famiglini, P. Palma, A. Cappiello, Rapid, hydrolysis-free, dilute-and-shoot method for the determination of buprenorphine, norbuprenorphine and their glucuronides in urine samples using UHPLC-MS/MS, J. Pharm. Biomed. Anal. 166 (2019) 236–243. [28] M.T. Truver, M.J. Swortwood, Quantitative analysis of novel synthetic opioids, morphine and buprenorphine in oral fluid by LC-MS-MS, J. Anal. Toxicol. 42 (2018) 554–561. [29] Y. Liu, X. Li, A. Xu, A.F. Nasser, C. Heidbreder, Simultaneous determination of buprenorphine, norbuprenorphine and naloxone in human plasma by liquid chromatography/tandem mass spectrometry, J. Pharm. Biomed. Anal. 120 (2016) 142–152. [30] A. Polettini, M.A. Huestis, Simultaneous determination of buprenorphine, norbuprenorphine, and buprenorphine-glucuronide in plasma by liquid chromatography-tandem mass spectrometry, J. Chromatogr. B 754 (2001) 447–459. [31] S. Gopal, T.-B. Tzeng, A. Cowan, Characterization of the pharmacokinetics of buprenorphine and norbuprenorphine in rats after intravenous bolus administration of buprenorphine, Eur. J. Pharm. Sci. 15 (2002) 287–293.
[32] S. Pirnay, F. Hervé, S. Bouchonnet, Liquid chromatographic-electrospray ionization mass spectrometric quantitative analysis of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in rat plasma, J. Chromatogr. B 41 (2006) 1135–1145. [33] A. Joshi, B. Parris, Y. Liu, C. Heidbreder, P.M. Gerk, M. Halquist, Quantitative determination of buprenorphine, naloxone and their metabolites in rat plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, Biomed. Chromatogr. (2017). [34] H. Hoja, P. Marquet, B. Verneuil, Determination of buprenorphine and norbuprenorphine in whole blood by liquid chromatography-mass spectrometry, J. Anal. Toxicol. 21 (1997) 160–165. [35] D. Favretto, G. Frison, S. Vogliardi, Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor-buprenorphine in urine, blood and hair samples, Rapid Commun. Mass Spectrom. 20 (2006) 1257–1265. [36] C. Cohier, L. Chevillard, S. Salle, P. Risède, O. Roussel, B. Mégarbane, Editor’s Highlight, Neurorespiratory effects of buprenorphine and ethanol in combination: a mechanistic study of drug-Drug interactions in the rat, Toxicol. Sci. 155 (2017) 389–399. [37] M. Feinberg, Validation of analytical methods based on accuracy profiles, J. Chromatogr. A 1158 (2007) 174–183. [38] F.T. Peters, O.H. Drummer, F. Musshoff, Validation of new methods, Forensic Sci. Int. 165 (2007) 216–224. [39] Food and Drug administration, Guidance for Industry. Q2B Validation of Analytical Procedures: Methodology, 1996 (accessed 08/12/ 2019) http:// www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/ guidances/ucm073384.pdf. [40] Food and Drug administration, Guidance for Industry. Bioanalytical Method Validation, 2001 (accessed 08/12/ 2019) http://www.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107. pdf. [41] Commission, Commission decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, Official Journal of the European Communities 2002, 221, 8-35. http://cemu10.fmv.ulg.ac.be/ostc/2002657EC.pdf, 2002 (accessed 08/12/ 2019). [42] S.M.R. Wille, W. Coucke, T. De Baere, F.T. Peters, Update of standard practices for new method validation in forensic toxicology, Curr. Pharm. Des. 23 (2017) 5442–5454. [43] W. Lindner, I.W. Wainer, Requirements for initial assay validation and publication in J. Chromatography B, J. Chromatogr. B 707 (1998) 1–2. [44] E. Chapuzet, N. Mercier, S. Bervoas-Martin, B. Boulanger, P. Chevalier, P. Chiap, D. Grandjean, P. Hubert, P. Lagorce, M. Lallier, M.C. Laparra, M. Laurentie, J.C. Nivet, Chromatographic dosage methods in biological mediums : a validation strategy. Report of an SFSTP commission, STP Pharma. Pratiques 7 (1997) 169–194. [45] E. Rozet, A. Ceccato, C. Hubert, E. Ziemons, R. Oprean, S. Rudaz, B. Boulanger, P. Hubert, Analysis of recent pharmaceutical regulatory documents on analytical method validation, J. Chromatog. A 1158 (2007) 111–125. [46] D.E. Moody, J.D. Laycock, A.C. Spanbauer, D.J. Crouch, R.L. Foltz, J.L. Josephs, L. Amass, W.K. Bickel, Determination of buprenorphine in human plasma by gas chromatography-positive ion chemical ionization mass spectrometry and liquid chromatography-tandem mass spectrometry, J. Anal. Toxicol. 21 (1997) 406–414. [47] K.A. Hadidi, J.S. Oliver, Stability of morphine and buprenorphine in whole blood, Int. J. Legal Med. 111 (1998) 165–167. [48] H. Jiang, Y. Wang, M.S. Shet, Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma, J. Chromatogr. B 879 (2011) 2663–2668. [49] Y. Liu, X. Li, A. Xu, Simultaneous determination of buprenorphine, norbuprenorphine and naloxone in human plasma by liquid chromatography/tandem mass spectrometry, J. Pharm. Biomed. Anal. 120 (2016) 142–152. [50] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS, Anal. Chem. 75 (2003) 3019–3030. [51] C.T. Viswanathan, S. Bansal, B. Booth, A.J. DeStefano, M.J. Rose, J. Sailstad, V.P. Shah, J.P. Skelly, P.G. Swann, R. Weiner, Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays, Pharm. Res. 10 (2007) 1962–1973. [52] E. Stokvis, H. Rosing, J.H. Beijnen, Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun. Mass Spectrom. 19 (2005) 401–407. [53] T. Berg, D.H. Strand, 1 3 C labelled internal standards–a solution to minimize ion suppression effects in liquid chromatography-tandem mass spectrometry analyses of drugs in biological samples? J. Chromatogr. A 1218 (2011) 9366–9374. [54] S. Wang, M. Cyronak, E. Yang, Does a stable isotopically labeled internal standard always correct analyte response? A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma, J. Pharm. Biomed. Anal. 43 (2007) 701–707. [55] C. Polson, P. Sarkar, B. Incledon, Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry, J. Chromatogr. B 785 (2003) 263–275.
C. Cohier, S. Salle, A. Fontova et al. / Journal of Pharmaceutical and Biomedical Analysis 180 (2020) 113042 ¨ [56] Restek. Colonnes LC c¨ ore-shellRaptor Biphenyl. https://clicktime.symantec. com/3LVnuhm5qWRJziAMCNKxaDf6H2?u=https%3A%2F%2Fwww.restek. fr%2Fcatalog%2Fview%2F40873 (accessed 08/12/ 2019). [57] Y. Chang, D.E. Moody, Effect of benzodiazepines on the metabolism of buprenorphine in human liver microsomes, Eur. J. Clin. Pharmacol. 60 (2005) 875–881.
[58] T. Seldén, M. Roman, H. Druid, LC-MS-MS analysis of buprenorphine and norbuprenorphine in whole blood from suspected drug users, Forensic Sci. Int. 209 (2011) 113–119. [59] P. Kintz, Deaths involving buprenorphine: a compendium of French cases, Forensic Sci. Int. 121 (2001) 65–69.
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