Accepted Manuscript

Title: Simplifying sample pretreatment: application of driedblood spot (DBS) method to blood samples, includingpostmortem, for UHPLC-MS/MS analysis of drugs of abuse

Author: Sara Odoardi Luca Anzillotti Sabina Strano-Rossi

PII: S0379-0738(14)00155-8DOI: http://dx.doi.org/doi:10.1016/j.forsciint.2014.04.015Reference: FSI 7574

To appear in: FSI

Received date: 7-10-2013Revised date: 26-3-2014Accepted date: 9-4-2014

Please cite this article as: S. Odoardi, L. Anzillotti, S. Strano-Rossi, Simplifyingsample pretreatment: application of dried blood spot (DBS) method to blood samples,including postmortem, for UHPLC-MS/MS analysis of drugs of abuse, Forensic ScienceInternational (2014), http://dx.doi.org/10.1016/j.forsciint.2014.04.015

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Simplifying sample pretreatment: application of dried blood spot (DBS) method to blood

samples, including postmortem, for UHPLC-MS/MS analysis of drugs of abuse

Sara Odoardi, Luca Anzillotti and Sabina Strano-Rossi

Forensic Toxicology Laboratory, Institute of public Health, Catholic University of Sacred Heart

L.go F. Vito, 1, 00168 Rome, Italy

*Corresponding Author: sabina.stranorossi@rm.unicatt.it; sabina.stranorossi@gmail.com;

Tel +39 0630156098; fax +39 063051168

Title Page (with authors and addresses)

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Graphical Abstract (for review)

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- Simple cadaveric blood samples preparation using DBS

- Determination of drugs of abuse in cadaveric blood

- Few microliters of sample required

- Forensic application

Highlights (for review)

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Abstract

The complexity of biological matrices, such as blood, requires the development of suitably selective

and reliable sample pretreatment procedures prior to their instrumental analysis. A method has been

developed for the analysis of drugs of abuse and their metabolites from different chemical classes

(opiates, methadone, fentanyl and analogues, cocaine, amphetamines and amphetamine-like

substances, ketamine, LSD) in human blood using dried blood spot (DBS) and subsequent

UHPLC–MS/MS analysis.

DBS extraction required only 100 µL of sample, added with the internal standards and then 3

droplets (30 µL each) of this solution were spotted on the card, let dry for 1 hour, punched and

extracted with methanol with 0.1% of formic acid. The supernatant was evaporated and the residue

was then reconstituted in 100 µL of water with 0.1% of formic acid and injected in the UHPLC–

MS/MS system. The method was validated considering the following parameters: LOD and LOQ,

linearity, precision, accuracy, matrix effect and dilution integrity. LODs were 0.05-1 ng/mL and

LOQs were 0.2-2 ng/mL. The method showed satisfactory linearity for all substances, with

determination coefficients always higher than 0.99. Intra and inter day precision, accuracy, matrix

effect and dilution integrity were acceptable for all the studied substances. The addition of internal

standards before DBS extraction and the deposition of a fixed volume of blood on the filter cards

ensured the accurate quantification of the analytes. The validated method was then applied to

authentic postmortem blood samples.

Introduction

Sample preparation, generally consisting of several steps, is the most time consuming part of

bioanalysis; it takes approximately 80% of the whole analysis time [1]. Conventionally, liquid–

liquid extraction [2-4], protein precipitation [5-7], solid-phase extraction [6-12] and solid-phase

microextrction [13] have been used as sample preparation techniques for blood.

Dried blood spot technique (DBS) was used for the first time on human blood in 1963 by Guthrie

and Susi for detection of phenylketonuria in large populations of newborn infants [14]. DBSs were

used not only in newborn screening for metabolic disorders but also in epidemiological [15],

toxicokinetics [16] and pharmacokinetic studies [17, 18], diagnostic screening [19-22] and

therapeutic drug monitoring [23-25].

This technique offers numerous advantages, for example a less invasive sampling method through

the possibility of collecting blood after a small finger or heel prick. Enhanced stability was

described for many analytes, for example cocaine, zopiclone and some benzodiazepines [26, 27]

on sampling cards at room temperature. Therefore samples could be easily stored and transported

without the need for refrigeration. Another advantage in using DBS is a lower biohazard risk to

handlers than liquid blood samples, reducing the infection risk of HIV/AIDS and other infectious

pathogens to a minimum. Furthermore, DBS deals with small blood volumes.

DBS's drawbacks are related to quantitative analysis. In fact quantification of the analytes of

interest is often based on internal standard method, but most of the DBS methods use to add internal

standards to solvent used to extract the spot [28-31]. Another drawback is that this methodology has

to be used in conjunction with analytical techniques capable of detecting the low amounts of

analytes present in few microliters of blood. In the past few years the improvement in the sensitivity

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of analytical instruments, especially tandem mass spectrometers, allowed for the quantification of

components extracted from DBS.

The aim of this study was to validate a UHPLC–tandem mass spectrometry method for the

simultaneous determination and quantification of illicit drugs and their metabolites using DBS on

postmortem blood samples. In our method, blood samples were mixed with internal standards prior

to spotting on the card in order to minimize spotting variability that could affect quantitative results.

The method was validated and applied to authentic blood specimens from autopsy cases.

Standards and reagents

Methanolic solutions (1 mg/mL) of amphetamine, amphetamine-D5, methamphetamine,

methamphetamine-D5, methylenedioxyamphetamine (MDA), MDA-D5,

methylenedioxymetamphetamine (MDMA), MDMA-D5, methylenedioxyethylamphetamine

(MDEA), MDEA-D5, benzoylecgonine (BEG), BEG-D3, ecgonine methyl ester (EME), EME-D3,

cocaine, cocaine-D3, cocaethylene, cocaethylene D3, methadone, methadone-D3, morphine,

morphine-D3, O-6-monoacetylmorphine (O-6-MAM), O-6-MAM-D3, ketamine, ketamine-D4,

norketamine, norketamine-D4, LSD, LSD-D3, 2-oxo-3hydroxy-LSD and fentanyl-D5, and

methanolic solutions (100 µg/mL) of alfentanyl, sufentanyl, fentanyl and nor-fentanyl were

obtained from LGC Standards (Milan, Italy).

Water, acetonitrile, formic acid and methanol were purchased from Sigma-Aldrich (Milan, Italy);

ammonium formate was from Agilent (Agilent Technologies, Santa Clara, CA, USA). Standard

compounds were stored according to supplier recommendations.

Internal standard mixture preparation

A mixture of internal standards containing amphetamine-D5, methamphetamine-D5, MDA-D5,

MDEA-D5, MDMA-D5, BEG-D3, cocaine-D3, cocaethylene D3, EME-D3, methadone-D3,

morphine-D3, O-6-MAM-D3, ketamine-D4, noketamine-D4, fentanyl-D5 and LSD-D3 at 1 µg/mL

was prepared by dilution of the proper amount of each standard solution in methanol and stored at -

20°C until use.

Sample preparation

Bond Elut Dried Matrix Spotting cards from Agilent (Agilent Technologies, Santa Clara, CA, USA)

were used for DBS analysis. 10 µL of Internal Standards mixture (1 µg/mL) was added to 100 µL

of whole blood. 3 droplets of this solution, each of 30 µL, were deposed on the card, forming 3

spots. The blood spots were allowed to dry for at least 2 hours at room temperature. Then a 3 mm

diameter disk was removed from the center of sample area of each spot using a manual punch and

the 3 spots were put in a tube with 900 µL of methanol/0.1% formic acid. After 1 hour the tube was

centrifuged at 4000 rpm for 5 minutes. The supernatant was evaporated to dryness under nitrogen

stream at room temperature, redissolved in 100 µL of water with 0.1% of formic acid and 10 µL of

the sample were injected into the UHPLC.

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UHPLC-MS/MS analysis

The UHPLC system was an Agilent 1290 Infinity system: binary pump with integrated vacuum

degasser, high performance well-plate autosampler and thermostated column compartment

modules. The detection system was an Agilent 6460 triple quadrupole mass spectrometer (Agilent

Technologies, Santa Clara,CA, USA) with the Jet Stream electrospray ionisation source. The

column used for this study was a superficially porous Kinetex C18 column (2.6 μm, 100×2.1 mm

from Phenomenex, Bologna, Italy).

Column temperature was set at 40 °C. Mobile phases were A: 5 mM ammonium formate containing

0.1% formic acid and B: methanol/acetonitrile, ratio of 1:1 with 0.1% of formic acid. The gradient

elution was as follows: phase A at 100% for 1 min and then increase of the organic phase from 0 to

10% in 0.1 min, ramp to 15% in 3 min, to 40% in 1.8 min, to 70% in 1.1 min and finally increased

to 100% of phase B in 2 min. After 2 min the column was led to the original ratio within 3 min to

enable its equilibration. The flow rate was set to 300 μL/min, and the eluate was introduced into the

mass spectrometer by means of ESI Jet Stream in positive mode. Optimal MS parameters chosen

were the following: capillary voltage was set to 4,000 V, the ion source was heated up to 350 °C

and nitrogen was used as nebulizing and collision gas at 12 L/min and 40 psi, respectively; EM

voltage was set to +800 V and nozzle voltage at 0 V. Determination of the optimal multiple

reaction monitoring transitions and respective collision energies for both quantifier and qualifier

ions of all compounds was carried out by consecutive injections analysis of the individual standards

at a concentration of 1 μg/mL, through a specific Agilent optimizer software (Mass Hunter

Optimizer). The chosen ion transitions, respective collision energies and retention times (Rt) of all

compounds and internal standards are scheduled in Table 1.

Validation

Calibration curves and quality controls (QCs) samples were prepared in three drug-free human

whole blood specimens by the addition of the appropriate amount of mixture of standard at 10

µg/mL to obtain the following concentration 2, 10, 50, 100, 500 ng/mL (0.2, 1, 5, 10 and 50 ng/mL

for alfentanyl, fentanyl, nor-fentanyl and sufentanyl) for calibration curves, and 2, 50 and 500

ng/mL ( 0.2, 5, and 50 for alfentanyl, fentanyl, norfentanyl and sufentanyl) for low, medium and

high QCs. The resulting spiked blood samples were subjected to the previously described pre-

treatment procedure and finally injected into the UHPLC–MS/MS system, on four different days.

The LOD was calculated at a concentration value giving a s/n ratio of >3 for all the transitions

considered for each substance while the limit of quantitation (LOQ) was the minimum

concentration giving a s/n of >10 for all the transitions and an acceptable precision (expressed as

coefficient of variation percentage (CV%), <20%) and accuracy (misured by percentage error (E%),

<20%). These parameters were studied using serial dilutions of the substances of interest both in

fresh blood from living subjects and in postmortem blood in triplicate using different samples,

analysed on four different days.

The linearity of the method for each compound was studied in the range from the LOQ of each

substance to 500 ng/mL (from LOQ to 50 ng/mL for alfentanyl, fentanyl, nor-fentanyl and

sufentanyl), performing triplicate analyses for each level. Calibration curves were built by linear

regression of the area ratio of each substance with the corresponding internal standard versus the

concentration of analyte. The weighting factor used was 1/x, in order to avoid the data at the high

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end of the calibration curve to dominate the calculation resulting in excessive error in the low

calibration range.

Intraday precision was assessed by replicate analyses of spiked samples in a single day; interday

precision was evaluated by determination of spiked samples on four days. Precision was measured

by calculating the relative standard deviation (RSD or %CV, SD divided by the mean and

multiplied by 100). Accuracy was determined for each concentration level as well, calculated by the

percent deviation from the nominal concentration.

Matrix effect was investigated by comparing the responses (in terms of peak areas) of positive

samples obtained spiking blank DBS after extraction with the analytes to those obtained injecting

neat solutions prepared at the same concentrations; absolute recoveries were determined by

comparing the signals of QC samples to those obtained spiking blank DBS after extraction at the

same concentrations, according to Matuszewski [32].

Selectivity was assessed by analyzing six different negative blood samples, three from living

subjects and three postmortem, submitted to DBS pre-treatment procedure and injected into the

UHPLC-MS/MS. The resulting chromatograms were checked for possible interferences from

endogenous components. The acceptance criterion was no interfering peak higher than an analyte

peak corresponding to its LOD.

Dilution integrity, the accuracy of quantification of a diluted sample, was evaluated by diluting five

samples containing 5000 ng/mL for all the analytes (500 ng/mL for alfentanyl, fentanyl nor-

fentanyl and sufentanyl) with blank blood to achieve the concentration of 100 ng/mL (10 ng/mL for

alfentanyl, fentanyl, norfentanyl and sufentanyl) with 1:50 dilution. After the dilution, the internal

standards were added and the samples were extracted as described previously.

Identification criteria

The criteria to be fulfilled for the identification of analytes were Rt, the presence of three transitions

and their relative ion intensities. Rt should not vary more than ±2 %; relative ion intensities should

not vary more than ±10% for ions with relative intensities of >50%, ±20% for ions with relative

intensities between 10% and 50% and ±50% for ions with relative intensities of <10% [33].

Method application

The method developed was applied to 10 postmortem blood samples in order to determine the

eventual presence of illicit drugs. 100 µL of each blood sample were added with the internal

standards, extracted using the DBS technique and analysed by UHPLC-MS/MS.

RESULTS AND DISCUSSION

The method showed to be suitable for the analysis of drugs of abuse pertaining to different classes

on blood samples. Figure 1 shows a positive blood sample spiked at the concentration of 50 ng/mL

for all the analytes studied and at 5 ng/mL for alfentanyl, fentanyl, nor-fentanyl and sufentanyl.

The method was fully validated, demonstrating its suitability, and was therefore applied to authentic

postmortem blood samples.

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Validation Results

The relationships between analytes concentrations and peak area ratios was linear over the range 2–

500 ng/mLfor all analytes and from 0.2 ng/mL to 50 ng/mL for alfentanyl, fentanyl, nor-fentanyl

and sufentanyl. The linear determination coefficients (r2) values were higher than 0.99 for all

analytes.

The limits of detection (LODs), based on the calculation of signal-to-noise ratios equal to 3, were

0.5 ng/mL for all analytes except for EME (1 ng/mL), and for alfentanyl, fentanyl, nor-fentanyl and

sufentanyl (0.05 ng/mL), both for blood and postmortem blood. The limits of quantification (LOQs)

corresponded to the first calibration point at 2 ng/mL for all the analytes and 0.2 ng/mL for

alfentanyl, fentanyl, nor-fentanyl and sufentanyl (Table 2).

Table 3a schedules the mean repeatability, accuracy, recovery and matrix effect results obtained at

three different concentrations (low, medium and high) in three different blood samples.

As shown in Table 3a, the method presented satisfying intraday and interday precision and

accuracy. The recovery was assessed by comparing the peak area ratios of extracted and control QC

samples at three concentration levels as described before. The reason for this relative low recovery

could be the irreversible binding of the analytes to the filter paper. Despite the low values obtained

the proposed method could reach values of LOD and LOQ sufficiently low to be suitable for

forensic purposes by the use of a very sensitive technique as UHPLC/MS-MS.

The proposed analytical procedure allowed to obtain an acceptable matrix effect, calculated by

comparing areas obtained from blank DBS samples spiked with analytes after extraction and areas

from samples prepared at the same concentration in mobile phase A. The mean ionic suppression,

evaluated in three different blood samples, was in fact < 40% for all the analytes also at low

concentrations.

Table 3b shows the mean repeatability, accuracy, matrix effect, and recoveries obtained by

analysing three different postmortem samples spiked at three concentrations each (low medium and

high). The results were analogous to those obtained from fresh blood; matrix effect was in fact

always lower than 50% also at low concentrations, demonstrating the suitability of the method for

the analysis also of postmortem blood.

The selectivity of the method was acceptable, as no interfering peaks were observed analyzing

blank blood specimens from 6 different sources.

The dilution integrity of the method was proved by the ability to accurately quantify samples

containing high concentrations of analytes diluted 1:50 with percent error <20 on five experiments.

Method application on authentic postmortem blood samples

Ten postmortem blood samples were analyzed with the proposed method.

Four of them were positive for drugs of abuse. Case 1 was positive morphine (270 ng/mL), O-6-

MAM (49 ng/mL), BEG (37 ng/mL) and cocaine (2.4 ng/mL). Case 2 and 3 revealed the presence

of morphine, respectively at concentration of 2000 ng/mL and 500 ng/mL. In case 4 methadone was

detected at 750 ng/mL.

In figure 2 is reported, as an example, the extracted ionic chromatogram of case 1.

Blood samples were pre-treated with a method used routinely in our laboratory, consisting of a LLE

with 3 ml of ethyl acetate at pH 9. The analytes were detected and their quantitative results were

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compared with those obtained with the DBS method and were always in good agreement, showing

the same results with a variation in quantitative determination always lower than 10 %.

The main advantage of DBS with respect to the classic liquid/ liquid extraction is that DBS allows

for very simple sample pretreatment with the use of minimal amounts of solvents

Conclusions

A simple, fast and reliable procedure for the simultaneous determination of illicit drugs from

different chemical classes in blood samples was developed, based on DBS extraction and UHPLC–

MS/MS analysis. In the study here presented, the accurate quantification of the analytes was

ensured by collecting a fixed volume of blood on the filter cards and by adding internal standards

before spotting blood on to the filter paper. This approach also assured to overcome all those factors

that could affect the quantitative results during the whole analytical procedure. Only 100 μL of

blood were used for the analysis. The method validation showed good linearity, accuracy and

precision, and an acceptable matrix effect. Although recoveries were low, the method achieved

LODs and LOQs compatible with the forensic purposes of the analyses. This method was therefore

successfully applied to various postmortem blood samples.

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Figures captions

Figure 1: Extracted Ionic Chromatogram from a positive blood sample spiked at a concentration of

50 ng/mL (alfentanyl, fentanyl, norfentanyl and sufentanyl 5 ng/mL)

Figure 2: Extracted Ionic Chromatogram from authentic postmortem blood sample extracted by

DBS, showing morphine at 270 ng/mL, O-6-MAM at 49 ng/mL, cocaine at 2.4 ng/mL and BEG at

37 ng/mL.

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Figure 1

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Figure 2

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Table 1. LC-MS/MS parameters for the selected analytes and internal standards

( Rt: retention time; Q1: precursor ion mass; Q3: product ion masses; CE: collision

energy)

Compund IS Rt(min) Q1 (amu) Q3 (amu) CE (V)

2 oxo-3OH LSD LDS D3 5.6 356.1 237.0 25 313.0 25 222.1 35

Alfentanyl Fentanyl D5 7.3 417.1 197.1 20 268.1 20 314.1 20

Amphetamine Amphetamine D5 4.0 136.1 91.1 17

119.1 5 65.1 41

Amphetamine D5 / 4.0 141.1 124.1 5 93.0 33

BEG BEG D3 6.0 290.1 168.1 17 105.0 29 82.1 29

BEG D3 / 6.0 293.1 171.1 17

Cocaethylene Cocaethylene D3 7.0 318.2 196.1 17 82.1 21 77.0 61

Cocaethylene D3 / 7.0 321 199 20 85 20

Cocaine Cocaine D3 6.6 304.2 182.1 17 82.1 21 77.0 61

Cocaine D3 / 6.6 307.1 185.1 17 85.1 21

EME EME 1.0 200.0 182.1 16 82.1 24 65.1 40

EME D3 / 1.0 203.0 185.1 16 85.1 18

Fentanyl Fentanyl D5 7.3 337.2 105.1 41 132.1 33 188.1 21

Fentanyl D5 / 7.3 342.0 140.4 20 123.3 20

Ketamine Ketamine D4 5.9 238.1 124.9 26 220.0 10 179.0 14

Ketamine D4 / 5.9 242.1 129.0 26

LSD LSD D3 7.0 324.2 223.1 17 281.2 17 197.1 17

Table 1

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LSD D3 / 7.0 327.7 226.6 17

MDA MDA D5 4.5 180.1 163.0 5 135.0 17 105.0 21

MDA D5 / 4.5 185.0 110.0 21

MDEA MDEA D5 5.6 208.1 163.0 5 135.0 17 105.0 21

MDEA D5 / 5.6 213.1 135.0 9

MDMA MDMA D5 4.8 194.1 163.0 9 105.0 25 77.0 45

MDMA D5 / 4.8 199.1 165 9 135 17

Methadone Methadone D3 7.8 310.2 265.1 9 105.0 29 91.0 29

Methadone D3 / 7.8 313.2 220 29

Methamphetamine Methamphetamine

D5 4.4 150.1

91.0 17 119.0 5 65.1 45

Methamphetamine D5 / 4.4 155.1 121.1 5

Morphine Morphine D3 2.8 286.2 152.0 60 157.1 41 128.1 60

Morphine D3 / 2.8 289.1 164.9 60

Nor-Fentanyl Fentanyl D5 6.0 233.2 84.1 13 55.1 37 29.1 60

Norketamine Norketamine D4 5.8 224.1 125.0 20 206.9 4 51.0 96

Norketamine D4 / 5.8 228.1 129.0 24 211.1 8

O-6-MAM O-6-MAM D3 4.8 328.2 165.1 41 152.0 60 58.1 29

O-6-MAM D3 / 4.8 331.2 165.1 42 193.1 26

Sufentanyl Fentanyl D5 7.6 387.2 355.1 20 238.1 20 206.0 20

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Table 2: Limits of detection and limits of

quantitation for the selected analytes. Compound LOD

(ng/mL) LOQ

(ng/mL) 2 oxo-3OH LSD 0.5 2

Alfentanyl 0.05 0.2

Amphetamine 0.5 2

BEG 0.5 2

Cocaethylene 0.5 2

Cocaine 0.5 2

EME 1 2

Fentanyl 0.05 0.2

Ketamine 0.5 2

LSD 0.5 2

MDA 0.5 2

MDEA 0.5 2

MDMA 0.5 2

Methamphetamine 0.5 2

Morphine 0.5 2

Nor-Fentanyl 0.05 0.2

Norketamine 0.5 2

O-6-MAM 0.5 2

Sufentanyl 0.05 0.2

Table 2

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Table 3a: Intra and inter day precision (expressed as CV%) and accuracy (E%), recovery (R%) and matrix effect (ME%) data for selected

analytes in blood samples

Compound 2 ng/mL 50 ng/mL 500 ng/mL

intraday Interday ME% R%

Intraday interday ME%

R%

intraday Interday ME% R%

CV% E% CV% E% CV% E% CV% E% CV% E% CV% E% 2 oxo-3OH LSD 14 13 14 13 83 31 3 1 15 -19 77 15 10 -4 14 -4 76 33 Amphetamine 13 -6 9 -7 76 31 5 3 2 4 64 18 0.3 -1 1 2 82 46 BEG 11 -10 4 -11 86 33 6 3 7 9 69 20 2 -2 3 1 89 44 Cocaethylene 14 -4 3 -11 85 29 4 5 13 4 72 11 3 -1 3 3 88 45 Cocaine 8 -9 13 0.4 89 16 5 4 7 0.2 75 12 2 -1 2 4 88 54 EME 8 -6 6 -5 62 32 1 3 2 3 70 25 1 -1 3 1 47 34 Ketamine 9 2 12 9 86 20 5 1 6 6 67 18 2 -1 4 3 82 45 LSD 13 3 9 -14 74 26 4 -1 4 8 74 10 2 1 2 3 74 24 MDA 14 6 14 -8 79 22 2 4 8 -4 67 20 2 0.1 1 3 72 47 MDEA 14 -4 11 -14 86 32 4 5 6 7 68 19 2 -1 4 1 86 50 MDMA 12 7 7 -0.2 67 26 5 5 3 1 71 19 1 -1 3 4 77 54 Methadone 10 8 9 7 65 18 3 5 5 3 61 15 3 -1 3 2 72 31 Methamphetamine 14 -6 4 13 86 28 2 4 3 2 63 22 1 -1 2 3 86 48 Morphine 8 -8 12 -10 75 25 4 3 10 9 85 16 2 -2 3 4 68 33 Norketamine 11 -14 2 -12 81 18 1 3 8 8 65 18 1 -2 2 5 78 41 O-6-MAM 15 5 13 14 73 46 4 -3 8 2 71 17 3 -1 6 2 88 44

0.2 ng/mL 5 ng/mL 50 ng/mL Intraday Interday

ME% R% intraday interday

ME% R% Intraday Interday

ME% R% CV% E% CV% E% CV% E% CV% E% CV% E% CV% E%

Alfentanyl 14 13 14 -4 89 29 2 4 2 -1 68 14 11 -6 10 5 87 52 Fentanyl 13 14 14 13 80 23 4 4 7 -1 68 14 0.4 -6 3 4 79 54 Sufentanyl 4 10 13 10 84 35 3 4 5 -3 69 22 2 -1 2 3 83 48 Nor-Fentanyl 14 8 7 -6 87 26 5 4 10 -4 74 21 1 -2 4 5 89 44

Table 3a

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Table 3b: Intra and inter day precision (expressed as CV%) and accuracy (E%), recovery (R%) and matrix effect (ME%) data for selected

analytes in postmortem blood samples

Compound 2 ng/mL 50 ng/mL 500 ng/mL

intraday Interday ME% R%

Intraday interday ME%

R%

intraday Interday ME% R%

CV% E% CV% E% CV% E% CV% E% CV% E% CV% E% 2 oxo-3OH LSD 5 12 6 13 63 16 14 7 14 9 70 27 4 2 5 3 56 20 Amphetamine 6 -9 8 -5 55 36 4 14 3 7 91 21 6 -2 4 2 82 32 BEG 13 -4 9 -2 66 29 5 9 7 5 65 53 6 -13 8 -5 66 41 Cocaethylene 14 -4 5 -5 50 16 3 11 5 8 75 41 5 -13 7 -4 65 45 Cocaine 13 8 9 14 63 57 11 13 9 2 64 48 4 -12 6 -3 65 45 EME 6 9 6 12 78 27 10 2 5 4 61 29 2 -8 5 -2 67 36 Ketamine 12 2 10 4 61 47 4 15 5 9 69 60 1 -14 4 2 69 58 LSD 13 1 9 13 66 13 7 15 6 7 62 38 2 -13 1 4 65 25 MDA 11 -11 8 -8 79 23 4 15 5 -1 97 42 1 -3 2 -2 84 47 MDEA 3 10 6 6 84 14 8 -10 9 8 81 23 13 1 4 5 72 49 MDMA 12 6 7 12 69 21 0.4 13 4 6 83 31 14 -2 5 1 84 47 Methadone 11 11 8 9 56 22 13 5 8 3 58 20 3 -11 7 2 65 15 Methamphetamine 3 -4 4 7 62 16 1 14 3 5 64 16 3 -3 4 3 51 33 Morphine 1 -5 7 11 49 17 5 5 4 6 70 28 0.3 -1 4 2 63 39 Norketamine 10 8 5 5 68 16 6 7 7 5 69 33 2 -5 5 -1 70 26 O-6-MAM 4 -13 8 11 71 28 10 13 9 4 87 35 2 -1 4 2 89 45

0.2 ng/mL 5 ng/mL 50 ng/mL Intraday Interday

ME% R% intraday interday

ME% R% Intraday Interday

ME% R% CV% E% CV% E% CV% E% CV% E% CV% E% CV% E%

Alfentanyl 13 2 11 6 78 25 5 13 4 8 76 20 12 -2 7 2 63 17 Fentanyl 14 -12 13 -8 67 14 14 -10 7 -5 76 19 5 -11 6 -5 67 15 Sufentanyl 12 -1 11 9 68 13 6 9 5 3 67 22 3 -14 4 2 73 18 Nor-Fentanyl 1 -4 7 -5 54 15 4 12 5 7 73 21 1 -3 6 2 77 23

Table 3b