Matrix effect in the analysis of drugs of abuse from urine with desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS) and desorption electrospray ionization-mass spectrometry (DESI-MS)

Analytica Chimica Acta 699 (2011) 73–80

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Matrix effect in the analysis of drugs of abuse from urine with desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS) and desorption electrospray ionization-mass spectrometry (DESI-MS) Niina M. Suni a , Pia Lindfors a , Olli Laine a,1 , Pekka Östman b , Ilkka Ojanperä b , Tapio Kotiaho a,c , Tiina J. Kauppila a , Risto Kostiainen a,∗ a

Division of Pharmaceutical Chemistry, University of Helsinki, P.O. Box 56, Helsinki FI-00014, Finland Hjelt Institute, Department of Forensic Medicine, University of Helsinki, P.O. Box 40, Helsinki FI-00014, Finland c Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, Helsinki FI-00014, Finland b

a r t i c l e

i n f o

Article history: Received 28 March 2011 Received in revised form 2 May 2011 Accepted 3 May 2011 Available online 12 May 2011 Keywords: Ambient mass spectrometry Desorption electrospray ionization Desorption atmospheric pressure photoionization Matrix effect Drug analysis

a b s t r a c t We have studied the matrix effect within direct analysis of benzodiazepines and opioids from urine with desorption electrospray ionization-mass spectrometry (DESI-MS) and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS). The urine matrix was found to affect the ionization mechanism of the opioids in DAPPI-MS favoring proton transfer over charge exchange reaction. The sensitivity for the drugs in solvent matrix was at the same level with DESI-MS and DAPPI-MS (LODs 0.05–6 ␮g mL−1 ) but the decrease in sensitivity due to the urine matrix was higher with DESI (typically 20–160-fold) than with DAPPI (typically 2–15-fold) indicating better matrix tolerance of DAPPI over DESI. Also in MS/MS mode, DAPPI provided better sensitivity than DESI for the drugs in urine. The feasibility of DAPPI-MS/MS was then studied in screening the same drugs from five authentic, forensic post mortem urine samples. A reference measurement with gas chromatography-mass spectrometry (GC–MS) (including pretreatment) revealed 16 findings from the samples, whereas with DAPPI-MS/MS after sample pretreatment, 15 findings were made. Sample pretreatment was found necessary, since only eight findings were made from the same samples untreated. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Ambient mass spectrometry (MS) is a rapidly growing field that provides fast and direct analysis of solid sample surfaces or liquid samples introduced on a suitable surface [1–4]. Different ambient ionization MS methods, such as desorption electrospray ionization (DESI) [5], direct analysis in real time (DART) [6], desorption atmospheric pressure chemical ionization (DAPCI) [7], and desorption atmospheric pressure photoionization (DAPPI) [8] have been successfully used in the direct analysis of compounds from various samples, such as body fluids [6,9], fruits, plant leaves [10], milk [11], banknotes [6], textiles [6,12], and pharmaceutical formulations [13,14], just to mention a few, without any sample pretreatment. Currently the most used ambient MS technique is DESI that employs a pneumatically assisted charged spray to desorb and ionize the analytes [5]. In most cases, the desorption/ionization in DESI

∗ Corresponding author. Tel.: +358 919 159 134; fax: +358 919 159 556. E-mail address: risto.kostiainen@helsinki.fi (R. Kostiainen). 1 Present address: Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, 00250 Helsinki, Finland. 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.05.004

occurs by so-called droplet pick-up mechanism where the charged droplets dissolve and desorb solid analytes from the surface [5,15], followed by emission of ions from the charged droplets in a similar process to conventional ESI [16]. DESI provides good sensitivity, down to pg–ng (fmol–pmol) range, for polar compounds [17,18] but the ionization efficiency for non-polar compounds may be poor [8,10]. Variations of DESI, such as reactive DESI [19] and desorption ionization by charge exchange (DICE) [20] have been developed for improving the ionization efficiency for non-polar compounds. DESI-MS has been used in many bioanalytical applications including direct analysis of urine [9,21–23], blood [24], skin [25], hair, plants [5], and bacteria [26]. DAPPI-MS is an ambient MS method, introduced in 2007 [8], which provides good sensitivity for polar as well as for completely non-polar compounds. In DAPPI, a heated jet of mixed nebulizer gas and spray solvent vapor desorbs solid analytes from the surface, after which the ionization of the analytes takes place through photoionization and gas-phase ion–molecule reactions similar to dopant-assisted APPI [27]. First, the photons emitted by a krypton discharge vacuum ultraviolet (VUV) lamp ionize the spray solvent producing either solvent radical cations (e.g. toluene, anisole) or protonated solvent molecules (e.g. acetone) in positive ion mode.

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Depending on the ionization energies (IEs) and the proton affinities (PAs) of the spray solvent and the analyte, the ionization of the analyte can take place either via charge exchange producing radical cations M+ of the analyte or via proton transfer producing protonated analyte molecules [M+H]+ [28]. DAPPI-MS has been applied to direct analysis of active ingredients from various matrices such as tablets [8,29], plants [29,30], blotter paper [29], soil [10], circuit board, orange peel, and brain tissue [30]. DAPPI-MS provides same sensitivity level (pmol–fmol) as DESI-MS for polar compounds but the sensitivity for non-polar compounds has been shown to be better with DAPPI [8,10]. The absence of sample pretreatment and separation in ambient MS methods, however, makes analysis prone to matrix effects [31] that may lead to significant background disturbances, decreased sensitivity and non-robust analysis due to contamination of the mass spectrometer. DESI-MS has been shown to be less susceptible to matrix effects than ESI-MS [9,32,33], but still, the sensitivity of DESI-MS is found to decrease in the analyzes of complex biological samples [21,22,24,34]. Studies that directly compare the matrix effects between different ambient MS techniques are limited [35], but it appears that the matrix effects depend on both the desorption and the ionization mechanisms [11,35,36]. Here, we have studied the effect of urine matrix on the analysis of drugs of abuse, benzodiazepines and opioids (Fig. 1), with DESI-MS and DAPPI-MS. Due to the better tolerance of DAPPI towards the urine matrix, a DAPPI-MS/MS method was developed for screening of the benzodiazepines and opioids in forensic, post mortem urine samples and the feasibility of the method, with and without a pretreatment step, was compared to the conventionally used GC–MS method.

2. Experimental 2.1. Reagents and solvents Nordiazepam, diazepam, codeine hydrochloride, codeine-d3, and morphine-d3 were purchased from Lipomed AG (Arlesheim, Switzerland), oxazepam and temazepam from Orion (Helsinki, Finland), morphine sulphate, oxycodone, and prazepam from Sigma Chemical Co. (St. Louis, MO, USA), tramadol hydrochloride from Nycomed Christiaens (Brussels, Belgium), temazepam-d5 and oxycodone-d3 from Cerillant (Round Rock, TX, USA), dibenzepin from Sandoz Pharmaceuticals Corp. (East Hanover, NJ, USA), Nmethyl-N-(trimethylsilyl)trifluoroacetamide from Machery-Nagel GmbH & Co KG (Duren, Germany), Na2 HPO4 ·2H2 O from Riedel-deHaen (Seelze, Germany), and ␤-glucuronidase (E. coli) from Roche Diagnostics GmbH (Mannheim, Germany). Water was purified with a Milli-Q Plus purifying system (Millipore, Molsheim, France). Methanol (HPLC grade) and acetone (HPLC grade) were purchased from Mallinckrodt Baker B.V. (Deventer, The Netherlands), toluene (HPLC grade), formic acid (98–100%), and propionic anhydride from Sigma–Aldrich (Steinheim, Germany), and anisole (≥99.9%) from Fluka Chemie GmbH (Buchs, Switzerland). Isopropanol (HPLC-grade, HiPerSolv) was ordered from VWR Prolabo (Leuven, Belgium).

A poly(methyl methacrylate) (PMMA) (Vink Finland, Kerava, Finland) sampling surface was used in DAPPI-MS and a poly(tetrafluoroethylene) (PTFE) (Vink Finland, Kerava, Finland) sampling surface in DESI-MS analysis. The PMMA sheet (thickness 5 mm) was cut into 2 cm × 4 cm pieces. The PTFE film (thickness 0.25 mm) was cut into 2 cm × 4 cm pieces and attached to a PMMA piece. All sampling plates had four sample spots and were used only once to avoid memory effects. On the sample spots, 1 ␮L of the sample was applied and left to dry. 2.3. Ion sources and mass spectrometry DAPPI-MS and DESI-MS measurements were carried out with an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) in positive ion mode with a scan range of 100–500 m/z. A nanospray stand (Proxeon Biosystems A/S, Odense, Denmark) was attached at the place of the conventional ion source and a capillary extension (Agilent Technologies, Santa Clara, CA, USA) at the place of the spray shield. In front of the capillary extension, the Nanospray stand housed the DAPPI or DESI ion source, described in detail in our earlier work [10]. The DESI ion source consisted of a grounded solvent delivery line connected to a syringe pump and a coaxial line for the nebulizer gas (nitrogen at 10 bar). Analysis conditions were optimized to maximize the signal intensities for the drugs. The DESI spray was directed at the sample spot using an impact angle of 45◦ , wetting the surface with a visible area of approximately 0.5 mm in diameter. The collection angle was less than 10◦ . The distance between the sprayer tip and the surface was 1–2 mm and the distance between the sample spot and the MS inlet (capillary extension at −5000 V) was 1–2 mm. The drying gas (nitrogen) flow rate was 4 L min−1 and the temperature 250 ◦ C. The DAPPI ion source consisted of a heated nebulizer microchip [10,37] for mixing and heating the spray solvent and the nebulizer gas (nitrogen). Parameters for the DAPPI microchip (heating power, gas flow rate, impact angle of the spray, and the distance of the sample from the mass spectrometer and from the microchip nozzle) that have been studied and optimized in previous studies [8,28], were adopted here. Other analysis conditions were optimized to maximize the signal intensities for the drugs. Spray solvent was introduced with a syringe pump at a flow rate of 10 ␮L min−1 . A heating power of 5 W and a nebulizer gas flow rate of 180 mL min−1 resulted in a vapor plume that heated the PMMA surface to approximately 300 ◦ C at an area of roughly 1.5 mm in diameter [8,28,38]. The heated vapor plume from the nozzle of the microchip, positioned at an approximately 45◦ angle with respect to the PMMA surface, was directed at the sample spot in front of the mass spectrometer. The collection angle was approximately 20◦ . The distance of the microchip nozzle from the surface was 3 mm and the distance of the sample spot from the MS inlet (capillary extension at −3500 V for the drugs in solvent and at −3200 V for the drugs in urine) was 2–3 mm. The drying gas (nitrogen) flow rate was 3.5 L min−1 and the temperature 200 ◦ C for the drugs in solvent and 240 ◦ C for the drugs in urine. A krypton discharge VUV lamp (Heraeus Noblelight, Cambridge, UK), producing photons of 10.0 eV and a minor amount of 10.6 eV, was positioned above the sample plate.

2.2. Sample preparation

2.4. Limits of detection (LODs)

Stock solutions of the drugs (1 mg mL−1 ) were prepared in ethanol (nordiazepam, diazepam, oxazepam, temazepam, tramadol, and codeine), methanol (oxycodone), or water (morphine). Working solutions (0.01–100 ␮g mL−1 ) were prepared in water–methanol (50:50, v/v) or blank urine. Forensic post mortem urine samples from the Department of Forensic Medicine (Hjelt Institute, University of Helsinki) were stored at +4 ◦ C.

LODs were measured for the drugs as a mixture in both solvent and in urine matrix. In MS mode, the LOD was defined as the concentration giving a spectrum with a signal-to-noise ratio (S/N) = 3 • for the most intensive ion of the drug (M+ or [M+H]+ as presented in Table 1). For overlapping m/z values of the drugs (diazepam [M+H]+ at 285, 13 C isotope at m/z 286 and 37 Cl isotope at m/z 287; morphine M+ at m/z 285, 13 C isotope at m/z 286, and [M+H]+ at m/z 286; and

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Fig. 1. Structures and monoisotopic molecular weights (MW) of the studied compounds. Benzodiazepines are in the top row and opioids in the bottom row.

oxazepam [M+H]+ at m/z 287), additional samples were prepared without diazepam. In MS/MS measurements, the most intensive • parent ion (M+ or [M+H]+ ) was isolated using a width of 2 m/z. In MS/MS mode, the LOD was defined as the lowest concentration giving a spectrum with the correct product ions as compared with the reference spectra measured from separate drug standard solutions (in solvent and in urine matrices).

in 0.5 mL of water–methanol (50:50, v/v). The same criteria, as in determining the LODs, were used in the identification of the drugs.

2.5. Screening the post mortem urine samples with DAPPI-MS/MS

For the GC–MS analysis of the benzodiazepines, the post mortem urine samples were hydrolyzed with ␤-glucuronidase for 2 h at 37 ◦ C. Internal standards (temazepam-d5 and prazepam) were added to the hydrolyzed samples. After a liquid–liquid extraction with ethyl acetate, the samples were evaporated to dryness and silylated with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for 30 min at 85 ◦ C. Electron ionization GC–MS (GC-EI-MS) was performed with a 5975C VL mass selective detector coupled with a 7890A GC and a 7683B injector (Agilent Technologies, Santa

The post mortem urine samples were analyzed with DAPPIMS/MS with and without a sample pretreatment step. The pretreatment step (for 1 mL of the samples) consisted of a hydrolysis with ␤-glucuronidase over night at 37 ◦ C and extraction with Isolute HCX-5 (100 mg) mixed-mode solid-phase extraction cartridges (International Sorbent Technology, Hengoed, U.K.) as described in reference [39]. The extracted drugs were dissolved

2.6. Screening the post mortem urine samples with gas chromatography-mass spectrometry (GC–MS) and gas chromatography-nitrogen phosphorus detector (GC-NPD)

Table 1 LODs (S/N = 3) for the drugs with DAPPI-MS and DESI-MS from solvent and urine matrices and with DAPPI-MS/MS and DESI-MS/MS from urine matrix. The increase in the LODs due to the urine matrix is calculated by dividing the LOD in urine by the LOD in solvent. Drug

DAPPI-MS LOD (␮g mL-1 )

DAPPI-MS/MS LOD (␮g mL-1 )

Ion (m/z)

Solvent

Ion (m/z)

Urine

Nordiazepam Diazepam Oxazepam Temazepam Tramadol Morphine Codeine Oxycodone

[M+H]+ (271) [M+H]+ (285) [M+H]+ (287) [M+H]+ (301) M+ • (263) M+ • (285) M+ • (299) M+ • (315)

0.07 0.07 0.2 0.1 0.1 3 0.3 0.1

[M+H]+ (271) [M+H]+ (285) [M+H]+ (287) [M+H]+ (301) [M+H]+ (264) M+ • (285) [M+H]+ (300) [M+H]+ (316)

1 0.5 90 1 0.2 50 0.5 1.5

Drug

DESI-MS LOD (␮g mL-1 ) Ion (m/z)

Nordiazepam Diazepam Oxazepam Temazepam Tramadol Morphine Codeine Oxycodone

+

[M+H] [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(271) (285) (287) (301) (264) (286) (300) (316)

Increase 14 7 450 10 2 17 2 15

Precursor ion (m/z)

Urine

[M+H]+ (271) [M+H]+ (285) [M+H]+ (287) [M+H]+ (301) [M+H]+ (264) M+ • (285) [M+H]+ (300) [M+H]+ (316)

0.6 0.08 0.1 0.075 0.7 70 0.5 0.1

DESI-MS/MS LOD (␮g mL-1 ) Solvent 0.05 0.1 0.2 0.07 0.07 6 0.25 0.5

Ion (m/z) +

[M+H] [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(271) (285) (287) (301) (264) (286) (300) (316)

Urine 5 5 100 10 6 100 40 10

Increase 100 50 500 143 86 17 160 20

Precursor ion (m/z) +

[M+H] [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(271) (285) (287) (301) (264) (286) (300) (316)

Urine 0.6 0.6 0.7 0.75 15 70 50 7.5

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Table 2 Findings from the forensic post mortem urine samples with DAPPI-MS/MS (X means positive identification) and GC–MS or GC-NPD (positive identification by means of concentration). Sample 1

Nordiazepam Diazepam Oxazepam Temazepam Tramadola Morphine Codeine Oxycodone

Sample 2

c (␮g mL−1 ) GC–MSa

Pretreated sample

20

X X

Untreated sample

Sample 3

c (␮g mL−1 ) GC–MSa

Pretreated sample

c (␮g mL

a

c (␮g mL−1 )GC–MSa Pretreated sample

Untreated sample

X X

220 0.93 18

Sample 4

Nordiazepam Diazepam Oxazepam Temazepam Tramadola Morphine Codeine Oxycodone

Untreated sample

−1

14

X

X

X

Sample 5 a

) GC–MS

Pretreated sample

Untreated sample

0.09 0.56 0.13 0.8

X

X X X

X

X

c (␮g mL−1 ) GC–MSa

Pretreated sample

1.84 0.09 4.25 2.38

X X X X

0.1 1.5 3.9

X X

Untreated sample X X

X X

GC-NPD.

Clara, CA). The capillary column was a DB-5MS (12 m × 0.2 mm i.d. with 0.33 ␮m film, Agilent Technologies, Santa Clara, CA). The GC was used in the splitless and constant flow mode with an injection volume of 2 ␮L, column flow of 1.5 mL min−1 , injector port temperature of 250 ◦ C, and transfer line temperature of 300 ◦ C. The oven temperature was initially held at 130 ◦ C for 0.5 min and then increased by 20 ◦ C min−1 to a final temperature of 320 ◦ C, which was held for 2 min. The mass detector was operated in selected ion monitoring (SIM) mode using three characteristic ions for each drug. For the GC–MS analysis of morphine and codeine, the post mortem urine samples were hydrolyzed with ␤-glucuronidase for 16 h at 46 ◦ C. Internal standards (codeine-d3 and morphine-d3) and 1 mL of 0.5-M Na2 HPO4 (pH 9) were added to the hydrolyzed samples. The pH of the samples was adjusted between 8.9 and 9.4 by adding approximately 175 ␮L of 0.1-M sodium hydroxide. After liquid–liquid extraction with butyl acetate, the samples were evaporated to dryness and derivatized with propionic anhydride for 30 min at 80 ◦ C. GC-EI-MS was performed with a 5975C VL mass selective detector coupled with a 6890N GC and a 7683B injector (Agilent Technologies, Santa Clara, CA). The capillary column used was a HP-5MS (12 m × 0.2 mm i.d. with 0.33 ␮m film, Agilent Technologies). The GC was used in the splitless and constant flow mode with an injection volume of 1 ␮L, column flow of 1 mL min−1 , injector port temperature of 260 ◦ C, and transfer line temperature of 300 ◦ C. The oven temperature was initially held at 95 ◦ C for 0.5 min and then increased by 40 ◦ C min−1 to a final temperature of 320 ◦ C, which was held for 2 min. The mass detector was operated in selected ion monitoring (SIM) mode using three characteristic ions for each drug. For the analysis of oxycodone, the urine samples were hydrolyzed with ␤-glucuronidase for 90 min at 55 ◦ C. 0.5 mL of 0.5 M KH2 PO4 (pH 6.2), internal standard (oxicodone-d3) and 0.3 mL 1 M TRIS-buffer (pH 11) were added to the hydrolyzed samples. After liquid–liquid extraction with butylacetate, the samples were centrifuged and the organic phase was transferred to vials. GC-EI-MS was performed with a 5975B mass selective detector coupled with a 6890N GC and a 7683B injector (Agilent Technologies, Santa Clara, CA). The capillary column used was a HB-5MS

(12 m × 0.2 mm i.d. with 0.33 ␮m film, Agilent Technologies, Santa Clara, CA). The GC was used in the splitless and constant flow mode with an injection volume of 2 ␮L, column flow of 1.5 mL min−1 , injector port temperature of 250 ◦ C, and transfer line temperature of 300 ◦ C. The oven temperature was initially held at 120 ◦ C for 0.5 min and then increased by 30 ◦ C min−1 to a final temperature of 320 ◦ C, which was held for 2 min. The mass detector was operated in selected ion monitoring (SIM) mode using three characteristic ions for the drug. For the analysis of tramadol, internal standard (dibenzepin) and 150 ␮L of 1 M TRIS-buffer (pH 11) were added to the samples. After liquid–liquid extraction with butyl acetate, the samples were vortexed and the supernatants placed in vials. GC was performed with a 6890 GC and a 7683B injector (Agilent Technologies, Santa Clara, CA) with dual nitrogen phosphorus detector (NPD). The capillary columns used were HP-5 (15 m × 0.32 mm i.d. with 0.25 ␮m film, J&W Scientific) and DB-17 (15 m × 0.32 mm i.d. with 0.25 ␮m film, J&W Scientific, Folsom, CA). The GC was used in the splitless and constant flow mode with an injection volume of 1 ␮L, injector port temperature of 270 ◦ C, and detector temperature of 330 ◦ C. The oven temperature was initially held at 100 ◦ C for 0.4 min and then increased by 25 ◦ C min−1 to a temperature of 200 ◦ C. After that, the temperature was increased by 10 ◦ C min−1 to a temperature of 240 ◦ C, and after that increased by 25 ◦ C min−1 to a final temperature of 290 ◦ C, which was held for 10 min. 3. Results and discussion 3.1. Ionization of the drugs from solvent and urine matrices with DESI-MS and DAPPI-MS In DESI-MS, we studied seven different spray solvent compositions: water with 0.1% formic acid, water–methanol 50:50 (v/v), water–methanol 50:50 (v/v) with 0.1% formic acid, water–methanol 10:90 (v/v), water–isopropanol 50:50 (v/v) with 0.1% formic acid, water–isopropanol 50:50 (v/v), and water–isopropanol 10:90 (v/v). All the spray solvents studied produced protonated molecules of the drugs without adduct formation or fragmentation from both solvent and urine matrices. The pres-

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Fig. 2. DESI mass spectra of the drug mixture (a) in solvent matrix (1 ␮g mL−1 ) and (b) in urine matrix (50 ␮g mL−1 ). The 37 Cl isotopic peak of diazepam at m/z 287 overlaps with the [M+H]+ of oxazepam at m/z 287. With these concentrations, morphine was not detected in solvent or urine and oxazepam is not detected in urine.

ence of acid in the spray solvent improved the ionization efficiency of the drugs. Water–methanol 50:50 (v/v) with 0.1% formic acid provided best sensitivity for the drugs and was chosen for further measurements (mass spectra under the chosen conditions are presented in Fig. 2). In DAPPI, we studied acetone, toluene, anisole, and toluene–anisole 99.5:0.5 (v/v) mixture as the spray solvents. The benzodiazepines were ionized by proton transfer with all the studied spray solvents from both solvent and urine matrices, indicating high proton affinities of the compounds. The opioids, however, were ionized either by proton transfer or charge exchange depending on the spray solvent and the sample matrix. Acetone as the spray solvent produced only protonated drug molecules from both solvent and urine matrices. This is because acetone forms only protonated reagent molecules excluding the charge exchange reaction and the formation of M+• [8,29], a phenomenon known also in APPI [27,40]. With toluene, the opioids in the solvent matrix

•

appeared as M+ and [M+H]+ but when the matrix was urine, only abundant [M+H]+ were formed. With anisole, the opioids in the solvent matrix were detected predominantly as M+ but when the matrix was changed to urine, protonated molecules dominated the mass spectra, except in the case of morphine which remained • as a M+ The ionization of the drugs with toluene–anisole 99.5:0.5 (v/v) mixture was similar to with anisole only, as recognized also in earlier studies with APPI [41]. In contrast to acetone, toluene and anisole form radical cations (m/z 92 and m/z 108, respectively) as reagent ions which can react through charge exchange with the analytes that have IEs below those of toluene (8.83 eV [42]) or anisole (8.20 eV [42]) or through proton transfer if the PA of the analyte is higher than that of benzyl radical (PA = 831,4 kJ mol−1 [42]) or methoxyphenyl radical (PA ≈ 884 kJ mol−1 [43]) [28]. The PAs of the benzodiazepines are obviously higher than 884 kJ mol−1 , since they formed only abundant [M+H]+ . For the opioids in solvent matrix, the charge exchange reaction was clearly favored

Fig. 3. DAPPI mass spectra of the drug mixture (a) in solvent matrix (1 ␮g mL−1 ) and (b) in urine matrix (5 ␮g mL−1 ). The mass spectra of morphine (10 and 100 ␮g mL−1 in • solvent and in urine, respectively) shown in the inserts were measured separately, since the M+ of morphine overlaps with the [M+H]+ of diazepam. In addition, the 37 Cl isotopic peak of diazepam at m/z 287 overlaps with the [M+H]+ of oxazepam at m/z 287. However, oxazepam was not detected from the urine matrix with this concentration.

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Fig. 4. DAPPI product ion spectra of temazepam [M+H]+ (m/z 301) from (a) blank urine, (b) standard in solvent (1 ␮g mL−1 ), (c) standard in urine (1 ␮g mL−1 ), (d) pretreated post mortem urine sample 5, (e) untreated post mortem urine sample 5. The product ions (m/z 283 and m/z 255) of temazepam are circled.

over proton transfer with toluene, anisole and toluene–anisole 99.5:0.5 (v/v) mixture as the spray solvents. However, when the matrix was urine, proton transfer was the dominating ionization reaction (expect for morphine with anisole and toluene–anisole 99.5:0.5 (v/v)). Similarly, the ionization of the opioids was affected by the urine matrix of authentic post mortem urine samples: using toluene–anisole 99.5:0.5 (v/v) as the spray solvent, the opioids appeared mainly as [M+H]+ (except for morphine) from the untreated urine samples but after sample pretreatment by solid phase extraction, the opioids appeared again as M+ (Fig. 4). This means that the components of the urine matrix desorbed to the gas phase, most probably urea or its reaction products, can change the reagent ion composition and ionization mechanism in DAPPI. No fragmentation of the drugs was observed with any of the spray solvents studied in DAPPI. Toluene–anisole 99.5:0.5 (v/v) mixture was chosen as the spray solvent for further measurements • since it provided good sensitivity and instead of both M+ and • + + + [M+H] it produced mainly only either M or [M+H] . The spectra of the drugs in solvent and in urine matrices with toluene–anisole 99.5:0.5 (v/v) as the spray solvent are presented in Fig. 3 (the spectra with acetone, toluene, and anisole are presented in Appendix A, Figs. A.1 and A.2). The DAPPI mass spectra of morphine shown in the inserts of Fig. 1, S1 and S2 were measured separately since • morphine M+ overlapped with the diazepam [M+H]+ . 3.2. Matrix effect in DESI-MS and DAPPI-MS To investigate the matrix effect, we compared the LODs measured for the drugs both in solvent and in urine matrices between DESI-MS and DAPPI-MS. The LODs for the drugs in the solvent

matrix were at the same range, typically 0.05–0.5 ␮g mL−1 , with DESI and DAPPI (Table 1). The LODs for the drugs in urine matrix, however, were increased typically 20–160-fold with DESI and 2–15-fold with DAPPI, as compared to the LODs for the drugs in solvent matrix. This means that the matrix compounds, most likely salts and urea, which exist at high concentrations in urine, interfere with both DESI- and DAPPI-MS analysis. However, the matrix effect was clearly higher with DESI than with DAPPI. Parallel results have been reported in earlier studies showing that APPI is less susceptible to matrix effects than ESI, both in terms of ion suppression [44–48] and higher background [46,49,50]. The decrease in sensitivity with DESI due to urine matrix has been reported also in several earlier works [21,22,34]. The reason for the better matrix tolerance of DAPPI over DESI lies within the different desorption/ionization mechanisms. In DAPPI, the desorption process is thermal and only volatile or semivolatile compounds are efficiently evaporated to the gas phase [8,28]. Urine contains high concentrations of salts that are known to interfere with the ionization (the transition of the ions from liquid to gas phase) in MS [31,51,52]. In DAPPI, the salts in the urine samples are not efficiently evaporated from the sampling surface and thus don’t significantly interfere with the ionization. In DESI, however, the desorption likely occurs by a droplet pick-up mechanism [5,15] and salts may be co-desorbed with the analytes within the charged droplets. This may cause not only ion suppression in the ion emission process, but also high background and contamination of the atmospheric pressure ion source and the mass spectrometer. After analyzing urine samples with DESI-MS, we noticed a precipitation formed from the urine matrix components at the inlet of the mass spectrometer. With DAPPI, we noticed no such effect and thus, DAPPI enabled longer operation of the mass spectrometer than DESI with-

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out cleaning the inlet of the mass spectrometer. However, with both methods, the contamination of the ion source after analyzing large batches of urine samples was a problem. To visualize the desorption of the urine matrix in DESI and DAPPI, the desorbed urine was collected on a filter paper and imaged with a microscope. Crystals derived from desorbed salts of the urine matrix, were seen on the filter paper from the DESI experiment whereas the filter paper from DAPPI experiment appeared blank (see Appendix A). The LODs measured for the drugs in urine with DAPPI-MS/MS and DESI-MS/MS are presented in Table 1. The repeatabilities of the methods were up to 50% (n = 4) and thus the LODs need to be considered within these variations. The LODs with DAPPIMS/MS are typically 10 times lower than with DESI-MS/MS, like are the LODs from urine in MS mode. The poor sensitivity of DAPPI and DESI for morphine might be explained by the fact that morphine is the only compound having an acidic group; it may therefore partly exist as a negative ion in the gas phase, decreasing the sensitivity in positive ion mode. For the other drugs, the LODs in urine with DAPPI-MS/MS (0.1–1 ␮g mL−1 ) are within the concentration range of the drugs typically found in urine [53]. These results indicate that DAPPI-MS/MS is suitable for screening of the drugs in forensic urine samples. However, the LODs for benzodiazepines and opioids from urine with LC–MS and LC–MS/MS typically range from low ng/mL level to low ␮g/mL level [39,54–56] providing about one-two orders of magnitude better sensitivity as compared with DAPPI. 3.3. Screening of the drugs from post mortem urine samples with DAPPI-MS/MS Due to the better matrix tolerance and lower LODs for the drugs in urine with DAPPI, we studied the feasibility of DAPPI-MS/MS in screening of the benzodiazepines and opioids from five authentic, forensic post mortem urine samples and compared the results with a validated and conventionally used GC–MS method (for tramadol GC-NPD). In DAPPI-MS/MS, the urine samples were analyzed both untreated and also after glucuronide hydrolysis and SPE purification; for the GC–MS method the urine samples were analyzed after glucuronide hydrolysis, purification, and derivatization. Of the five samples, 16 findings were made with the GC–MS and GCNPD, 15 findings with DAPPI-MS/MS after the sample pretreatment and eight findings with DAPPI-MS/MS without the sample pretreatment (untreated samples) (Table 2). The small number of the findings from the untreated samples could be explained by the fact that the drugs studied are excreted in urine mainly as glucuronides [53], which were not monitored in the DAPPI-MS/MS analysis. On the other hand, the glycosidic bond of the glucuronides is relatively weak and could be dissociated into an aglycone (the parent drug) and glucuronic acid in the DAPPI process. For example, the dissociation of morphine glucuronide has been reported in DART-MS analysis [57]. In the mass spectra of the post mortem urine samples we could not see glucuronide conjugates of the drugs. This, however, might result from not only the dissociation of the glucuronide conjugate but also from inefficient desorption and ionization of the glucuronides with DAPPI. Sample pretreatment (including glucuronide hydrolysis) significantly improved the identification with DAPPI-MS/MS. The background disturbances were decreased providing improved specificity and therefore more reliable identification as demonstrated in Fig. 4 that presents an example of DAPPI-MS/MS measurements of temazepam from blank urine (a), standard in solvent (b), standard in urine (c), untreated post mortem urine sample (d), and pretreated post mortem urine sample (e). Some differences, however, were observed between the findings made with DAPPI-MS/MS after sample pretreatment and with GC–MS. Morphine in samples 3 and 5 and nordiazepam in sample 4 were not detected with DAPPI-MS/MS due to insufficient

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sensitivity. Findings made with DAPPI-MS/MS but not with GC–MS include temazepam in the pretreated sample 1 and oxazepam in the pretreated sample 2. Yet the findings made with DAPPIMS/MS after sample pretreatment correlated clearly better with the findings made with GC–MS than with the findings made with DAPPI-MS/MS without the sample pretreatment. Example spectra of the positive findings from the pretreated post mortem urine samples with DAPPI-MS/MS, together with the reference spectra of the standards from solvent and urine matrices showing the product ions used in the MS/MS identification of the drugs, are presented in Appendix A. 4. Conclusions DESI-MS and DAPPI-MS provide fast analysis with easy operation and a possibility to avoid time-consuming sample preparation. The sensitivity of DESI-MS and DAPPI-MS is comparable for polar compounds in simple (such as solvent) matrices. From a biological matrix urine, however, DAPPI provides approximately ten times better sensitivity for benzodiazepines and opioids and longer operation without the need to clean the mass spectrometer indicating better matrix tolerance of DAPPI over DESI. However, in DAPPI, the urine matrix may affect the ionization mechanism. These differences can be explained by the different desorption/ionization mechanisms of DESI and DAPPI. In both methods, however, the urine matrix decreased the sensitivity and cleaning of the ion source after analyzing large numbers of urine samples was necessary. Despite the good matrix tolerance of DAPPI-MS/MS, its performance in screening the drugs from forensic, post mortem urine samples was acceptable only after sample pretreatment. Yet even with sample preparation the feasibility of DAPPI-MS in the analysis of drugs from urine is not as good as that of GC–MS or LC–MS because of lower sensitivity, selectivity, and repeatability. The results in this study suggest that although many ambient MS methods have been shown to be powerful and fast direct analysis tools in various cases, it should always be carefully considered and studied whether the sample pretreatment steps can be bypassed without decreasing the performance of the method, especially in cases where large numbers of biological samples must be analyzed. Besides the matrix effects on the analysis quality, the contamination of the ion source and the mass spectrometer is a concern. Acknowledgements Academy of Finland, Instrumentarium Foundation, and the National Doctoral Programme in Nanoscience are acknowledged for financial support and Vink Finland for providing the PMMA sheets. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2011.05.004. References [1] R.M. Alberici, R.C. Simas, G.B. Sanvido, W. Romao, P.M. Lalli, M. Benassi, I.B.S. Cunha, M.N. Eberlin, Anal. Bioanal. Chem. 398 (2010) 265–294. [2] D.J. Weston, Analyst 135 (2010) 661–668. [3] M. Huang, C. Yuan, S. Cheng, Y. Cho, J. Shiea, Annu. Rev. Anal. Chem. 3 (2010) 43–65. [4] H. Chen, B. Hu, X. Zhang, Chin. J. Anal. Chem. 38 (2010) 1069–1088. [5] Z. Takáts, J.M. Wiseman, B. Gologan, R.G. Cooks, Science 306 (2004) 471–473. [6] R.B. Cody, J.A. Laramée, H.D. Durst, Anal. Chem. 77 (2005) 2297–2302. [7] Z. Takáts, I. Cotte-Rodríguez, N. Talaty, H. Chen, R.G. Cooks, Chem. Commun. (2005) 1950–1952. [8] M. Haapala, J. Pól, V. Saarela, V. Arvola, T. Kotiaho, R.A. Ketola, S. Franssila, T.J. Kauppila, R. Kostiainen, Anal. Chem. 79 (2007) 7867–7872.

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