Journal of Chromatography B 1171 (2021) 122625
Available online 6 March 2021
1570-0232/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Short communication
A sensitive LC-MS/MS method for the quantitation of oxycodone,
noroxycodone, 6
α
-oxycodol, 6β-oxycodol, oxymorphone, and
noroxymorphone in human blood
Michael T. Truver
a,*, Gerd Jakobsson
a,b, Maria D. Cherm`a
b, Madeleine J. Swortwood
c,
Henrik Gr´een
a,b, Robert Kronstrand
a,baDivision of Drug Research, Department of Biomedical and Clinical Sciences, Faculty of Medicine, Link¨oping University, 581 83 Link¨oping, Sweden bDepartment of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 587 58 Link¨oping, Sweden
cDepartment of Forensic Science, College of Criminal Justice, Sam Houston State University, Huntsville, TX, USA
A R T I C L E I N F O Keywords: Pharmacokinetics Oxycodone 6α-oxycodol 6β-oxycodol A B S T R A C T
The objective of this study was to develop and validate a highly sensitive method for the detection of oxycodone, noroxycodone, 6β-oxycodol, 6α-oxycodol, oxymorphone, and noroxymorphone in blood by liquid
chromatog-raphy tandem mass spectrometry. The analytes were extracted from blood (0.5 mL) using Bond Elut Certify Solid Phase Extraction columns, evaporated to dryness and reconstituted before analysis was performed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD. Academy Standards Board Standard Practices for Method Development in Forensic Toxicology were used for the validation of this method. The limit of quantitation for all analytes was established at 0.5 ng/mL. Calibration range for noroxymorphone, oxymorphone, 6α-oxycodol and
6β-oxycodol was 0.5–25 ng/mL and 0.5–100 ng/mL for noroxycodone and oxycodone. Precision (2.90–17.3%) and bias studies resulted in a ±15% deviation. There were no interferences observed from internal standard, matrix, or common drugs of abuse. Stability of all analytes at two concentrations at 24, 48, and 72 h in the autosampler did not exceed ±20% difference from the initial T0. Dilution integrity at a ten-fold dilution was acceptable as analyte concentrations ranged between (±18%) of the target concentration. Once validated, the method was used in a pilot dosing study of one male subject after taking a 10 mg immediate release tablet of oxycodone. Blood samples were collected at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 9, and 24 h after ingestion. Oxycodone and noroxycodone both reached Tmax at 1.5 h and had Cmax values of 25.9 and 12.8 ng/mL, respectively. Oxycodone, 6α-oxycodol, and 6β-oxycodol were detectable up to 9 h, while noroxymorphone and
noroxycodone were still detected at 24 h.
1. Introduction
Oxycodone is an opioid commonly encountered in forensic casework due to high potential of abuse for its heroin-like effects. Due to the extensive metabolism of oxycodone, interpretation issues can arise in forensic casework. Recently, in a study by Jakobsson et al, postmortem blood samples (n = 192) that contained concentrations of oxycodone were analyzed and investigated to determine if the inclusion of metab-olites (noroxymorphone, noroxycodone, and oxymorphone) and use of metabolite ratios could assist in resolving interpretation issues [1]. The study also discussed interpretation issues from naloxone as it shares a metabolite with oxycodone (nornaloxone also known as
noroxymorphone). It was concluded that concentrations above 0.2 µg of oxycodone/gram whole blood were likely to have contributed to toxicity and that low noroxycodone/oxycodone ratios could indicate acute intake with fatal outcome.
Understanding the pharmacokinetics of oxycodone and its metabo-lites is beneficial for the interpretation of oxycodone concentrations in forensic case work. In Cajanus et al, plasma concentrations of oxycodone and its metabolites were determined from breast cancer surgery patients after they were satisfied with their titrated intravenous dose of 1–3 mg of oxycodone [2]. There were a total of 1000 women in the study and oxycodone concentrations determined for 938 with geometric mean concentrations for oxycodone, noroxycodone, oxymorphone, and
* Corresponding author.
E-mail address: Michael.Truver@liu.se (M.T. Truver).
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noroxymorphone were 33.3, 1.46, 0.11, and 0.26 ng/mL, respectively. When the patient requested more analgesia, the mean concentration for oxycodone was 21.7 ng/mL.
Plasma concentrations for oxycodone and metabolites were deter-mined after 16 subjects were administered a 15 mg dose of oxycodone in Lalovic et al [3]. Metabolites in this study included noroxycodone, oxymorphone, noroxymorphone, α-noroxycodol, β-noroxycodol, α
-oxy-morphol, β-oxy-oxy-morphol, α-oxycodol, and β-oxycodol. Oxycodone,
nor-oxycodone, oxymorphone, noroxymorphone, α-oxycodol, and
β-oxycodol achieved Tmax at 1.08, 1.22, 0.98, 1.55, 1.55, and 3.03 h,
respectively. Oxycodone and noroxycodone had the most abundant concentrations found in this study, while concentrations of metabolites such as the oxycodols were low and comparable to each other.
Whole blood and oral fluid samples were collected from healthy subjects (n = 12) after being administered a 20 mg controlled release oxycodone tablet in Cone et al [4]. Samples were collected up to 52 h and concentrations in whole blood and oral fluid for oxycodone and its metabolites were determined. For blood, Tmax for oxycodone,
norox-ycodone, and noroxymorphone was 4.8, 4.8, and 3.8 h, respectively and corresponding Cmax values were 20.6, 15.6, and 6.4 ng/mL. Although
oxymorphone was included in method, there were no detectable con-centrations observed in the study which applied a lower limit of quan-titation (LLOQ) of 5 ng/mL.
Improved detection of oxycodone use has been accomplished by the inclusion of metabolites, sensitive instrumentation, and optimized extraction methods [3,5–9]. In Al Asmari et al, whole blood samples were analyzed by liquid chromatography mass spectrometry (LC-MS/ MS) for the quantitation of opioids including oxycodone, noroxycodone, and oxymorphone [7]. The group utilized Bond Elut C-18 solid phase extraction (SPE) cartridges for extraction, which helped reach LLOQs of 1 ng/mL for oxycodone, noroxycodone, and oxymorphone. Neuvonen et
al used LC-MS/MS for the quantitation of oxycodone, noroxycodone,
oxymorphone, and noroxymorphone in human plasma [8]. Low LLOQs of 0.1 ng/mL for oxycodone and oxymorphone and 0.25 ng/mL for noroxycodone and noroxymorphone were achieved using Oasis MCX SPE cartridges for extraction.
In Cone et al, as authors found that establishing a lower LLOQ may improve the detection of analytes in blood and the detection of minor metabolites such as oxycodols, thus improving interpretation [4]. Based on these findings we used a previously validated method as basis for the development and validation of a method with increased sensitivity for the detection of oxycodone, noroxycodone, 6β-oxycodol, 6α-oxycodol,
oxymorphone, and noroxymorphone in blood by LC-MS/MS [1]. The increased sensitivity and inclusion of minor metabolites will be benefi-cial for the application of this method to a human dosing experiment with oxycodone.
2. Materials and methods 2.1. Chemicals and reagents
Noroxymorphone (NOM), oxymorphone (OM), oxymorphone-d3
(OM-d3, deuterium atoms on methyl group of tertiary amine, isotopic
purity >99%), noroxycodone (NOC), noroxycodone-d3 (NOC-d3,
deuterium atoms on methoxy group, isotopic purity >98%) , oxycodone (OC) and oxycodone-d3 (OC-d3, deuterium atoms on methoxy group,
isotopic purity >99%) were obtained from Cerilliant (Round Rock, TX, USA). 6α-oxycodol (αOCL) and 6β-oxycodol (βOCL) were purchased
from Cayman Chemical (Ann Arbor, MI, USA). Acetonitrile, ammonia solution (25%), dichloromethane, formic acid, glacial acetic acid, iso-propanol, and sodium acetate trihydrate were purchased from Merck (Darmstadt, Germany). Bond Elut Certify SPE columns (130 mg, 10 mL cartridge, 120 µm particle size) were purchased from Agilent Technol-ogies (Santa Clara, CA, USA). A Milli-Q system was used for purified water. Evaporation was performed on a TurboVap® LV Automated Solvent Evaporation System from Biotage (Uppsala, Sweden).
2.2. Solid phase extraction
Mixed methanolic stock solutions were prepared to create calibrators at 0.5, 1.0, 2.5, 5.0, 10, and 25 ng/mL for NOM, OM, αOCL, βOCL, and at
0.5, 5.0, 10, 25, 50 and 100 ng/mL for NOC and OC when fortified in blood. Internal standard stock solution was prepared with all three deuterated internal standards at 200 ng/mL in methanol. Internal standard (25 uL) was added to 0.5 mL of blood to achieve the final concentration of 10 ng/mL. Standard or quality control solutions (25 μL)
were then added, followed by 2 mL of 0.1 M acetate buffer (pH 6). The buffered blood was then centrifuged (4000g, 10 ◦C) for 10 min. Bond
Elut Certify columns (consisting of a nonpolar C8 and a strong cation- exchange sorbent) were conditioned with 2 mL methanol, 2 mL deion-ized water, and 2 mL 0.1 M acetate buffer (pH 6). The buffered blood was then loaded to the column and washed with 2 mL deionized water, 2 mL 0.1 M acetate buffer (pH 4), and 2 mL methanol then dried for 5 min. The compounds were eluted with 2 mL dichloromethane:iso-propanol (80:20) to which 2% of ammonium hydroxide was added and evaporated under nitrogen at 40 ◦C. Samples were reconstituted in 100
μL of mobile phase A.
2.3. Instrumentation
Analytes were analyzed on an Acquity UPLC® I-class (Waters Corp., Milford, MA, USA) coupled to a Waters Xevo TQD. Separation was achieved on an Acquity HSS T3 column (1.7 µm, 2.1 × 100 mm), held at 30 ◦C. The gradient of 0.001% formic acid in 10 mM ammonium formate Table 1
Optimized parameters for oxycodone, noroxycodone, 6β-oxycodol, 6α-oxycodol, oxymorphone, noroxymorphone, and deuterated internal standards.
Compound name Precursor (m/z) Product (m/z) Cone (V) Collision (V) Retention Time (min) Transition Ratio Internal Standard
OC 316.2 241.0 34 32 4.14 1.73 OC-d3 316.2 256.1 34 30 NOC 302.1 187.0 32 24 4.01 1.21 NOC-d3 302.1 227.0 32 28 βOCL 318.2 199.1 34 34 3.82 0.83 OM-d3 318.2 256.1 34 30 αOCL 318.2 199.1 34 34 3.62 1.75 OM-d3 318.2 256.1 34 30 OM 302.1 242.1 40 28 3.06 0.37 OM-d3 302.1 227.0 40 30 NOM 288.1 213.0 32 28 2.90 2.25 OM-d3 288.1 184.1 32 46 OC-d3 319.1 244.0 40 32 4.11 NOC-d3 305.1 190.0 36 24 3.99 OM-d3 305.1 230.0 36 30 3.05
(pH 5.2, A) and 0.001% formic acid in acetonitrile (B) was held at 2% B for 1.5 min then increased to 25% over 4.7 min, increased to 95% B and held for 1 min, decreased to 2% B and re-equilibrated for 1 min. Data were acquired and analyzed using Waters MassLynxTM software (Wa-ters, version 4.1 SCN 940). Two transitions per analyte and one transi-tion per internal standard were optimized as seen in Table 1. Concentrations were determined using area ratios of the analyte and internal standard plotted against the added concentration. Source pa-rameters consisted of cone and capillary voltage at 41 V and 0.39 kV, respectively and desolvation gas and gas flow were 500 ◦C and 1000 L/
hr, respectively.
2.4. Method validation
The method was validated according to the American Academy of Forensic Sciences (AAFS) Standards Board (ASB) Standard Practices for Method Development in Forensic Toxicology [10]. Parameters such as calibration model, precision, bias, carryover, dilution integrity, stability, limit of detection (LOD), LOQ, matrix effects, recovery, process effi-ciency, and interferences were evaluated for the method validation. Calibration model was evaluated using 6 non-zero calibrators over 5 days and plotting residuals. Precision and bias were determined at low, medium, and high concentrations in triplicate over 5 runs. Low con-centrations were 3 times the lowest calibrator (1.5 ng/mL), medium concentrations were 10 ng/mL for NOM, OM, αOCL, and βOCL, 40 ng/
mL for NOC and OC, and high concentrations were 20 ng/mL for NOM, OM, αOCL, and βOCL, 80 ng/mL for NOC and OC (80% of the highest
calibrator). Bias was deemed acceptable if it was within ±20% of each concentration. Precision (CV) included the study of intraday and inter-day precision with the largest % reported. The acceptable limit for both precision studies was ±20%. Carryover was assessed by analyzing a negative blank matrix (internal standard only) after the highest
calibrator over three different runs. Dilution integrity was assessed by performing a 10 times dilution on the highest calibrator in triplicate and considered acceptable within ±20% of the target concentration. Sta-bility was determined in the autosampler (5ºC) at low and high con-centrations analyzed in triplicates. The time intervals investigated were 24, 48, and 72 h in the autosampler and were deemed acceptable within ±20% of initial standard area/internal standard ratio. LOQ and LOD were determined using three different negative matrix sources fortified at the lowest calibrator concentration and analyzed in triplicate over three runs. LOQ were evaluated for acceptable precision and bias (±20%), transition ion ratios (±20%), and signal to noise (>10). Matrix effects, recovery, and process efficiency were determined using 10 different negative matrix sources at low (1 ng/mL) and high concen-trations (10 ng/mL and 80 ng/mL) and were considered acceptable within ±25% of the target area at the respective concentration. Calcu-lations of these parameters performed as mentioned in Matuszewski et al
[11]. Interference studies included investigation of potential in-terferences from internal standards, common drugs of abuse, and matrix and were considered an interferant if greater than the LOD.
2.5. Pilot study
One subject (male) was administered a single immediate release 10 mg tablet of Oxycodone Actavis. Blood samples were collected at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 9, and 24 h after ingestion. The Swedish Ethical Review Authority approved this study (Dnr 2020- 00102) and written consent was obtained from the subject.
3. Results and discussion 3.1. Method validation
The primary goal of this study was to develop and validate a sensitive method for the quantitation of oxycodone, noroxycodone, 6β-oxycodol, 6α-oxycodol, oxymorphone, and noroxymorphone in blood samples
from a single dose study. In Table 2, validation results such as precision, bias, and dilution integrity are shown. Interday precision ranged from 2.90 to 12.5% and intraday precision ranged between 4.13 and 17.3%, both precision studies were within the ±20% and deemed acceptable. Bias ranged from − 15.0 to 6.93% for all analytes and determined to be acceptable as it was well within the ±20%. After performing the ten-fold dilution, analyte concentrations were between 82 and 106% of the target concentration and therefore dilution integrity of a ten times dilution was deemed acceptable.
Previous methods have established the need for low LLOQs in order to improve the detection of minor metabolites such as OM, αOCL, and
βOCL in samples from a dosing study [2,4]. The LLOQ for the current method was determined to be 0.5 ng/mL for all analytes after analyzing the lowest calibrator in three different sources in triplicate (n = 9) and ensuring that identification criteria such as transition ratios and reten-tion time as well as bias were within acceptable limits. The LOD was
Table 2
Validation results for oxycodone, noroxycodone, 6β-oxycodol, 6α-oxycodol, oxymorphone, and noroxymorphone.
Analyte LLOQ (ng/mL) ULOQ (ng/mL) Interday Precision (%CV) Intraday Precision (%CV) Bias (%) Dilution Integrity (%) Lowa Midb Highc Lowa Midb Highc Lowa Midb Highc
OC 0.5 100 6.14 3.22 2.90 6.66 3.90 4.13 6.05 5.70 1.82 106 NOC 0.5 100 5.62 3.24 4.11 5.65 4.40 6.09 1.25 2.22 0.92 105 βOCL 0.5 25 11.3 8.19 6.70 11.5 8.22 9.85 −5.38 − 5.13 − 2.56 92 αOCL 0.5 25 4.45 5.23 4.86 5.62 5.81 9.84 −11.4 − 15.0 − 9.62 82 OM 0.5 25 6.96 5.25 5.15 10.2 4.96 5.38 1.71 − 12.4 − 11.7 100 NOM 0.5 25 10.3 11.4 12.5 14.0 12.3 17.3 −0.25 − 0.66 6.93 94
aLow concentration: 1.5 ng/mL for all analytes.
b Middle concentration: 10 ng/mL for NOM, OM, αOCL, and βOCL, 40 ng/mL for NOC and OC. cHigh concentration: 20 ng/mL for NOM, OM, αOCL, and βOCL, 80 ng/mL for NOC and OC.
Table 3
Mean matrix effect, recovery, and process efficiency for oxycodone, norox-ycodone, 6β-oxycodol, 6α-oxycodol, oxymorphone, and noroxymorphone in
blood.
Analyte Matrix Effects (%, n =10)
Recovery (%, n =
10) Process Efficiency (%, n =10) Lowa Highb Lowa Highb Lowa Highb
OC −4 − 1 86 79 83 78 NOC 0 − 4 78 81 77 78 βOCL −4 3 79 82 76 85 αOCL 1 2 83 82 84 84 OM −16 − 16 74 73 62 61 NOM −33 − 24 58 62 39 47 OC-d3 −5 − 6 86 77 82 72 NOC-d3 −4 − 12 78 83 74 73 OM-d3 −16 − 25 78 75 65 56
aLow concentration: 1 ng/mL for all analytes.
b High concentration: 20 ng/mL for NOM, OM, αOCL, and βOCL, 80 ng/mL for NOC and OC.
administratively set at 0.5 ng/mL (the lowest calibrator) due to the detection of compounds not being beneficial to a pharmacokinetic study without quantifiable concentrations. The upper limit of quantitation (ULOQ) for NOM, OM, αOCL, and βOCL was 25 ng/mL and 100 ng/mL
for NOC and OC. After reviewing residual plots, the calibration model for NOM, OM, αOCL, and βOCL was determined to be quadratic with 1/x
weighting, while NOC and OC were determined to be linear with 1/x weighting. No carryover was determined in the negative blank matrix injected after the high calibrator as no analyte signal >LOD was detec-ted. There were no interferences observed after analyzing 10 different negative matrix samples and no significant interferences (>LOD)
observed from analyzing only the internal standards. Additionally, there were no interferences detected after analyzing common drugs of abuse (opioids, stimulants, benzodiazepines) and common over the counter drugs (ibuprofen, acetaminophen, salicylic acid). Stability of all analytes at both concentrations and at all three time points (24, 48, and 72 h) in the autosampler did not exceed ±20% difference from the initial T0 and
therefore was deemed acceptable.
Matrix effects, recovery, and process efficiency results are shown in
Table 3. All analytes were within the allotted ±25% for matrix effects except for NOM at the low concentration of 1 ng/mL (− 33%). Deuter-ated internal standards were comparable to their non-deuterDeuter-ated
counterparts, ensuring mitigation of suppression observed. NOM had the lowest recovery and process efficiency as well, while all other ana-lytes had >70% recovery and process efficiency. Although NOM underperformed, it was deemed acceptable for this validation as it did not compromise its performance in LLOQ, precision, and bias studies.
3.2. Pilot study
Chromatograms from the low QC, negative sample, and the 1.5 h sample from the study are shown in Fig. 1. Blood concentrations for OC and its metabolites from the subject were determined and time- concentration profiles were constructed. OC and the major metabolite from this study, NOC, are shown in the Fig. 2. OC was detectable up to 9 h, while NOC was detectable up to the last collection point at 24 h. Tmax
for OC and NOC was observed at 1.5 h for both analytes, with a Cmax of
25.9 and 12.8 ng/mL, respectively. In Cone et al, OC and NOC both reached Tmax at 4.8 h and the OC concentration (20.6 ng/mL) was
slightly higher than the NOC concentration (15.6 ng/mL) for Cmax [4].
These results were similar to the current study’s findings, and the higher Tmax determined in Cone et al can be attributed to the controlled release
formulation administered.
The minor metabolites from this study (NOM, αOCL, βOCL) are
shown in Fig. 2. Tmax for NOM, αOCL, βOCL was 5, 2, and 4 h,
respec-tively. Cmax for NOM, αOCL, βOCL was 2.27, 2.13, and 1.56 ng/mL,
respectively. The longer Tmax for βOCL when compared to αOCL was also
observed in Lalovic et al [3]. The oxycodols were detectable for 9 h, while NOM was detectable up to 24 h. Similar to Cone et al, there were no detectable concentrations of unconjugated OM found in this study even with the improved LOQ of 0.5 ng/mL [4]. This is in accordance with Lalovic et al that reported that OM had the lowest concentration of all metabolites measured in their study due to its conjugation with glucuronic acid to form oxymorphone-3ß-D-glucuronide [3]. The improved sensitivity from this method ensured detection of minor metabolite concentrations and thus improved the detection window of NOC.
4. Conclusion
In conclusion, a sensitive method for the quantitation of oxycodone, noroxycodone, 6β-oxycodol, 6α-oxycodol, oxymorphone, and
norox-ymorphone in blood was validated. This method was applied to samples from a pilot dosing study and the results were comparable to previous
studies. It was determined that this method is suitable for future phar-macokinetic studies involving the administration of oxycodone.
Funding
This work was supported by the Strategic Research Area in Forensic Sciences at University of Link¨oping, Sweden [Grant numbers 304399 and 304774].
CRediT authorship contribution statement
Michael T. Truver: Funding acquisition, Data curation,
Methodol-ogy, Validation, Writing - original draft, Formal analysis. Gerd
Jakobsson: Funding acquisition, Data curation, Methodology,
Valida-tion, Writing - review & editing, Formal analysis. Maria D. Cherm`a: Writing - review & editing, Supervision. Madeleine J. Swortwood: Funding acquisition, Writing - review & editing. Henrik Gr´een: Funding acquisition, Writing - review & editing. Robert Kronstrand: Funding acquisition, Conceptualization, Data curation, Methodology, Validation, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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