ORIGINAL PAPER

UHPLC-ESI-MS/MS method for direct analysis of drugsof abuse in oral fluid for DUID assessment

Sabina Strano-Rossi & Luca Anzillotti &Erika Castrignanò & Marialinda Felli &Giovanni Serpelloni & Roberto Mollica &

Marcello Chiarotti

Received: 11 March 2011 /Revised: 29 April 2011 /Accepted: 13 May 2011 /Published online: 15 June 2011# Springer-Verlag 2011

Abstract An ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry method forthe direct analysis in oral fluid (OF) of several abused drugs andmetabolites in a single chromatographic run was set up andvalidated. Amphetamine, methamphetamine, morphine, O-6-monoacetylmorphine, cocaine, codeine, methylenedioxy-methamphetamine (MDMA), methylenedioxyethylamphet-amine, methylenedioxyamphetamine, methadone,benzoylecgonine (BEG), Δ9-tetrahydrocannabinol (THC),ketamine, and cocaethylene were determined in a singlechromatographic run with no sample pretreatment, afteraddition of the respective deuterated internal standards. Themethod was designed to perform a confirmation analysis onthe residual OF samples after the preliminary on-site screeningtest, and it was applied on preservative buffers from differentdevices (Mavand Rapidstat, Concateno DDS, and GreinerBio-One) or on neat OF samples. The method was suitable tobe applied to the small amounts of sample available for theconfirmatory analysis after the preliminary on-site screeningor on undiluted OF samples. Limits of detection varied from 5(morphine) to 0.2 ng/mL (methamphetamine, MDMA, BEG,and cocaethylene). The method was linear for all thesubstances involved, giving quadratic correlation coefficientsof >0.99 in all the different preservative buffers checked. In

addition, repeatability and accuracy were satisfactory for themajority of the substances, except for a few cases. Thedeveloped method was subsequently applied to 466 residualsamples from on-site screening performed by police officers.Of these samples, 74 showed the presence of cocaine andmetabolites; THC was detected in 49 samples. Two samplesshowed codeine and morphine while MDMAwas detected in11 samples and ketamine in four samples.

Keywords Forensic toxicology. Oral fluid .

UHPLC-MS/MS . DUID

Introduction

Driving under the influence of drugs (DUID) is a majorproblem worldwide. The assessment of drug- and alcohol-impaired driving is, therefore, a challenge of primaryimportance. Oral fluid (OF) has proved to be a biologicalmatrix with potential applications in road-side drug screen-ing due to its correlation to plasma time courses of drugappearances/disappearances [1–9]. OF can, therefore, be analternative specimen as a confirmation sample for toxico-logical analysis. Furthermore, it is an easily availablemedium that can be collected with noninvasive methods,without the intrusion of privacy and achieved undernonmedical staff supervision. It can be collected underdirect observation, thereby reducing or eliminating prob-lems such as adulteration and substitution that are oftenassociated with urine collection.

In Italy, since August 2010, the law considers OF as analternative biological specimen for the determination ofDUID [10]. The law allows that impaired drivers can bepreliminarily screened using on-site tests; nowadays, thelaw states a presence/absence criterion and no cut-off

S. Strano-Rossi (*) : L. Anzillotti : E. Castrignanò :M. Felli :M. ChiarottiInstitute of Legal Medicine, Università Cattolica del Sacro Cuore,L.go F. Vito 1,00168 Rome, Italye-mail: sabina.stranorossi@rm.unicatt.it

G. Serpelloni : R. MollicaDepartment of Antidrug Policies,Presidency of the Council of Ministers,Via Po 16/A,00198 Rome, Italy

Anal Bioanal Chem (2011) 401:609–624DOI 10.1007/s00216-011-5108-y

values or minimum required performance levels arerecommended, neither for the screening nor for confirma-tion analyses (zero tolerance).

Many rapid on-site OF tests have been developed in thelast years due to the high attention in most countries todrug-impaired driving. To be used for forensic purposes,screening results must anyway be confirmed by a chro-matographic spectrometric technique: identity confirmationof forensically relevant compounds, such as drugs of abuse,is a necessary step in medico-legal events such as controlsof people involved in DUID.

The possibility to directly analyze the residual OF samplefrom on-site screening was sought to analyze exactly the sameOF sample used for the screening, avoiding problems relatedto different times of sampling. Our goal was, therefore, to setup an ultra-high-performance liquid chromatography–electro-spray ionization–tandem mass spectrometry (UHPLC-MS/MS) method that could directly analyze the main abuseddrugs in a single chromatographic run and with a minimalsample pretreatment, due to the small amount of sampledisposable for the confirmatory analysis after the preliminaryon-site screening.

This is possible by the fact that some of the on-site kits,such as Concateno DDS (the previous Cozart Rapiscan) andMavand Rapidstat allow to store the remaining aliquot of thesample used for the screening; Draeger commercializes a“twin” OF collection device, DCD 5000, that allows thesimultaneous sampling of two aliquots of pure OF; other kits,on the contrary, do not allow to store the same OF aliquot:Securetec Drugwipe, for example, utilizes a few microliters ofOF for the screening analysis. Another device is, therefore,necessary for the subsequent OF collection for confirmatoryanalysis, such as the DCD 5000 or the new Greiner Bio-One(GBO) “Saliva collection kit.”

The present work was performed in the framework of aproject of the Italian Antidrug Policies Department. Theaim was to evaluate the performances of different kits, bythe direct confirmatory analysis of the same sample usedfor the screening test. This preliminary part of the projectinvolved the development of a simple, rapid, and reliableanalytical method to be applied on the confirmation ofresults—both positive and negative—from on-site tests, inorder to evaluate the analytical performances of differentOF on-site devices. The proposed method was, therefore,developed on neat OF and on OF diluted with buffers fromdifferent collection kits, in order to evaluate a wide range ofpossibilities for the collection and analysis of OF samples.The kits involved in the study were among those rated“promising” from the most recent literature and theprevious projects, mainly in terms of sensitivity and easilyat road-site (Rosita-2 and DRUID projects [11, 12]); someof them are those mentioned above. Experiments are still inprogress for the overall evaluation of the screening kits.

The analytical method was, therefore, set up for itsapplication to OF diluted with DDS, Rapidstat and GBOpreservative solutions and to pure OF samples, in case ofcollection of a further aliquot with the Draeger kit or analogcollection kits (e.g., Salivette device).

An advantage of liquid chromatography–tandem massspectrometry is the possibility to analyze aqueoussamples with the so-called dilute and shoot technique,which in practice involves a simple dilution of thesamples to be analyzed in addition to the isotopicdilution with the internal standards, if the instrumenthas sufficient sensitivity. Ultra-high-performance liquidchromatography makes direct analysis possible with veryhigh resolution and reduced run time. At the same time,the new electrospray ionization (ESI) Jet Stream tech-nology developed by Agilent (Agilent Technologies,Santa Clara, CA, USA) allows greater signal and reducednoise through a thermal gradient focusing technologythat uses super-heated nitrogen (N2) to improve iongeneration and desolvation.

A method for direct analysis of OF residual sample forvarious drugs of abuse from on-site tests was, therefore,developed using these technologies.

Materials and methods

Chemicals and reagents

Methanolic solutions (1 mg/mL) of amphetamine, am-phetamine D5, methamphetamine, methamphetamine D5,methylenedioxyamphetamine (MDA), MDA-D5, methyl-enedioxymetamphetamine (MDMA), MDMA-D5, meth-ylenedioxyethylamphetamine (MDEA), MDEA-D5, Δ9-tetrahydrocannabinol (THC), THC-D3, benzoylecgonine(BEG), benzoylecgonine D3 (BEG-D3), cocaine, cocaineD3, cocaethylene, codeine, codeine D3, methadone, metha-done D3, morphine, morphine D3, O-6-monoacetylmorphine(O-6-MAM), O-6-MAM-D3, ketamine, and ketamine D4were obtained from LGC Standards (Milano, Italy).

Water, acetonitrile, formic acid, and methanol werepurchased from Sigma-Aldrich (Milano, Italy); ammoniumformiate was from Agilent (Agilent Technologies, SantaClara, CA, USA). Standard compounds were storedaccording to supplier recommendations.

Blank samples collection

Ten drug-free OF samples were obtained from laboratorystaff and used for the preparation of calibration curves andfor the repeatability and matrix effect studies.

Blank OF samples were collected using Salivette devices(Sarstedt, Germany). Samples (1–2 mL) were collected

610 S. Strano-Rossi et al.

according to manufacturer’s specifications and were storedat −20 °C until analysis.

Evaluation of preservative buffers from commercialOF collection kits

Preservative buffers from Mavand Rapidstat kit (Arnika,Milano, Italy) and Concateno DDS kit (Concateno Italia,Pomezia, RM) were evaluated for their analysis in LC/MS.According to manufacturer specifications, OF collectedwith these devices is nearly 0.3 mL (Rapidstat, 0.25±0.03 mL; DDS, 0.34±0.03 mL [13]), which is diluted in 1±0.05 mL of unknown composition buffers. Also, the GBOSaliva collection kit (Greiner Bio-One, Kresmunster,Austria) was evaluated in laboratory for the direct LC-MS/MS analysis. This latter kit collects a mean of 4.1 mLof OF by rinsing the oral cavity with 4 mL of extractionsolution, containing citric acid [14].

Neat OF samples (blanks, calibrators or controls) were,therefore, diluted in the proper amount of preservative buffer inorder to compensate the dilution of real samples in these buffers.

On-site samples collection and analysis

Real OF samples were from on-site tests, performed bypolice officers on suspected drugged drivers and werecollected using two different kits that allowed samplestorage after preliminary screening test: DDS or Rapidstat.

OF samples were collected and analyzed with theimmunometric assays according to the kit specificationsand, after the screening, the remaining aliquot was sent tothe laboratory for the confirmation analysis.

Preparation of stock-positive OF samples

Individual methanolic stock solutions containing 1 mg/mL ofeach of the listed standards were used to prepare a mixture ofstandards at a concentration of 1 μg/mL (MIXDRUGS). Inthe same way, a mixture of deuterated analogs was prepared ata concentration of 1 μg/mL (MIXDRUGS-D3). Calibrationcurves were prepared by the addition of the appropriateamount of MIXDRUGS to 2 mL of blank OF to obtain thefollowing concentrations: 0.2, 0.5, 1, 2, 4, 5, 10, 25, 50, 100,and 200 ng/mL. Quality control samples (QC) were preparedat 10, 50, and 100 ng/mL, using a different working solution.Stock and working OF samples as well as methanolic standardsolutions were stored at −20 °C until use.

Calibration and QC samples preparation

Four different calibration curves were prepared to comparesamples collected from on-site testing with the availablecollection kits, as well as to study pure OF samples: neat OF,

OF diluted in DDS preservative buffer, OF diluted in Rapidstatpreservative buffer and OF diluted in GBO collecting solution.

Hence, 250 μL of each sample were added with 25 μLof MIXDRUGS-D3. The dilution of real samples in thesampling solutions was compensated through the dilutionof calibrators and QC samples with the appropriate amountof the respective preservative buffers, as also performed inother research papers [15–17]. Neat OF samples werediluted with 250 μL of mobile phase A.

Real samples preparation

Two hundred fifty microliters of real samples from on-sitetesting were only added with 25 μL of MIXDRUGS-D3. Ifthe total volume of sample was lower, the amount ofMIXDRUGS-D3 was added in a proportional amount.Twenty microliters of the solutions were then directlyinjected in the UHPLC-MS/MS equipment.

UHPLC-MS/MS equipment

The UHPLC system was an Agilent 1290 Infinity system:binary pump with integrated vacuum degasser, high-performance well-plate autosampler and thermostattedcolumn compartment modules.

The detection system was an Agilent 6460 triple quadru-pole mass spectrometer (Agilent Technologies, Santa Clara,CA, USA) with the Jet Stream electrospray ionisation source.

The column evaluated for this study was superficiallyporous: Kinetex C18 column (2.6 μm, 100×2.1 mm fromPhenomenex, Bologna, Italy).

UHPLC-MS/MS method

Column temperature was set at 40 °C, and injection volumewas 20 μL. Mobile phase A: 5 mM ammonium formiatecontaining 0.05% formic acid and mobile phase B:methanol/acetonitrile ratio of 1:1 with 0.1% of formic acidwere utilized with a gradient from 0% B to 100% B within8 min. Absolute acetonitrile, absolute methanol, andacetonitrile/methanol ratio of 90:10 and 80:20 were alsotested as mobile phase B. The flow rate was set to 500 μL/min, and the eluate was introduced into the massspectrometer 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 washeated up to 350 °C and nitrogen was used as nebulizingand collision gas at 12 L/min and 40 psi, respectively; EMvoltage was set to +800 Vand nozzle voltage at 0 V.

Three time segments were created: the first, completelydiscarded, started from 0 till 1.7 min with 100% phase A toelute the high concentration of unknown salts and preser-vatives contained in the buffer solutions of the devices

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in OF 611

analyzed; the second one, from 1.71 till 7.1 min, includedmost of the analytes; the third one was created to increasethe sensibility to THC. For THC analysis, in the thirdacquisition time segment, the nozzle voltage was set to2,000 Vand electromultiplier at +1,000 V. Determination ofthe optimal multiple reaction monitoring transitions andrespective collision energies for both quantifier and quali-fier ions of all compounds was carried out by consecutiveinjections analysis of the individual standards at a concen-tration of 1 μg/mL, through a specific Agilent optimizersoftware (Mass Hunter Optimizer). The chosen ion tran-sitions, respective collision energies and retention times(RT) of all compounds and internal standards (IS) arescheduled in Table 1.

Method validation

The analytical method was fully validated taking intoconsideration the following parameters [18]:

Specificity and assessment of matrix effect

Specificity of the method was studied by analyzing 20blank samples that were analyzed for assessment ofpotential interferences from endogenous substances. Spec-ificity had also been studied on buffer solutions ofcommercial devices. Interferences from OF and bufferswere considered not significant if at the retention times oftarget analytes were not detected interfering peaks. Speci-ficity of the method was further assessed by analyzingsamples spiked with high concentrations (500 ng/ml) ofsubstances with analog structure and with therapeuticdrugs. The following substances were studied: ibuprofen,nikethamide, nimesulide, ketoprofen, diclofenac, tramadol,fenbutrazate, amfepramone, caffeine, heptaminol, ephed-rine, phenylpropanolamine, cathine, pseudoephedrine,metoxyphenamine, fenfluramine, norfenfluramine, phen-metrazine, phendimetrazine, fentermine, benzphetamine,dextromoramide, ethylamphetamine, fencamine, fencanf-

Table 1 Characteristic transitions, collision energies and retention times

Target compound Transitions (m/z) Collision energy (V) RT (min)

Morphine 286.2→157.1, 152.0, 128.1 41 (157.1); 60 (152); 60 (128.1) 2.2

Morphine D3 289.1→164.9 60 2.2

Codeine 300.2→165.1, 128.0 45 (165.1); 60 (128.0) 2.9

Codeine D3 303.1→165.1 45 2.9

Amphetamine 136.1→119.1, 91.0, 65.1 5 (119.1); 17 (91.0); 41 (65.1) 3.0

Amphetamine D5 141.1→93.1, 124.1 33 (93.1); 5 (124.1) 3.0

Methamphetamine 150.1→119.0, 91.0, 65.1 5 (119.0); 17 (91.0); 45 (65.1) 3.25

Methamphetamine D5 155.1→121.1 5 3.25

MDA 180.1→163.0, 135.0, 105.0 5 (163.0); 17 (135); 21 (105) 3.3

MDA D5 185→110 21 3.3

MDMA 194.1→163.0, 105.0, 77.0 9 (163.0); 25 (105); 45 (77.0) 3.6

MDMA D5 199.1→165; 135 9(165); 17 (135) 3.6

O-6-MAM 328.2→165.1, 152.0, 58.1 41 (165.1); 60 (152); 29 (58.1) 3.65

O-6-MAM D3 331.2→193.1, 165.1 26 (193.1); 42 (165.1) 3.65

MDEA 208.1→163.0, 135.0, 105.0 9 (163); 21(135); 25 (105) 4.3

MDEA D5 213.1→135 9 4.3

Ketamine 238.1→220.0, 179.0, 124.9 10 (220); 14 (179); 26 (124.9) 4.9

Ketamine D4 242.1→129.0 26 4.9

BEG 290.1→168.1, 105.0, 82.1 17 (168.1); 29 (105.0); 29 (82.1) 5.1

BEG D3 293.1→171.1 17 5.1

Cocaine 304.2→182.1, 82.1, 77.0 17 (182.1); 21 (82.1); 61 (77.0) 5.7

Cocaethylene 318.2→196.1, 82.1, 77.1 17 (196.1); 33 (82.1); 60 (77.1) 6.1

Cocaine D3 307.1→185.1, 85.1 17 (185.1); 21 (85.1) 5.7

Methadone 310.2→265.1, 105.0, 91 9 (265.1); 29 (105); 29 (53) 6.8

Methadone D3 313.2→220 29 6.8

THC 315.2→193.1, 135.1, 123.0 18 (193.1); 18 (135.1); 30 (123) 7.4

THC D3 318.3→196.3 21 7.4

In bold are the internal standards (IS), and transitions set in italics are used for quantitation

612 S. Strano-Rossi et al.

amine, fenethylline, methylphenidate, meperidine, pentazo-cine, pentetrazol, clobenzorex, dimethylamphetamine, cro-tetamide, cropropamide, fenproporex, mefenorex,mephentermine, prolyntane, modafinil, oxycodone, alfen-tanyl, sufentanyl, and fentanyl.

Sufficient specificity was achieved if the peaks did notinterfere with the analyte of interest in terms of RT andcharacteristic transitions.

To assess any potential ion suppression/enhancementdue to the sample matrix or to the preservative buffersthree QC samples at 10, 50, and 100 ng/mL, prepared intriplicate in different sample matrix and in differentcollection buffers, were compared to the same concen-trations of pure standards in the mobile phase A. Thematrix effect was evaluated comparing the peaks areas ofthe analytes and of their respective deuterated IS inmatrix with those of the samples at the same concen-trations in mobile phase A. According to Matuszewski[19], the matrix effect (ME) was calculated as follows: ME%=B/A×100, being B the mean areas obtained in samplematrices and A the areas of the neat standards in mobilephase. In addition, calibration curves prepared by dilutingthe methanolic mixture of pure standards in the mobilephase Awere compared to those prepared in neat OF or inOF diluted with the different buffers. The slopes were thencompared by ANOVA.

Limits of detection and limits of quantitation

The LOD was figured out at a concentration value giving as/n of >3 for all the transitions considered for eachsubstance while the limits of quantitation (LOQ) was theminimum concentration giving a s/n of >10 for all thetransitions and an acceptable precision (coefficient ofvariation percentage (CV%), <20%) and accuracy (percent-age error (E%), <20%). These parameters were studiedusing scalar dilutions of the substances of interest in OF intriplicate, analyzed in three different days.

Linearity and dilution integrity

The linearity of the method for each compound wasstudied in the range from the LOQ of each substance to200 ng/mL, performing triplicate analyses for eachlevel. Calibration curves were built by linear regressionof the area ratio of each substance with thecorresponding internal standard versus the concentrationof analyte; the weighting factor used was 1/x. Individualoutlier points with accuracy differing more than 20%, ifpresent, were automatically eliminated by the quantita-tive software.

The dilution integrity was evaluated by diluting threesamples spiked at a concentration of 1,000 ng/mL (1:10)

and three samples at a concentration of 10,000 ng/mL(1:100) with blank OF. Dilution integrity was achievedif the samples could be quantified with a relative errorof <15% with respect to the nominal concentration.

Precision and Accuracy (analytical bias)

Precision was evaluated by replicate analysis (n=5) of QCOF samples at three different concentrations: 10, 50, and100 ng/mL on the same day (intra-day assay), andreproducibility was determined by replicate analysis (n=5)over 5 days in a 6-month period (n=25) of the sameconcentrations as for repeatability (inter-day assay). Preci-sion, expressed as CV% of the measured values, wasexpected to be less than 15% for all concentration levels(<20% near LOQ).

Accuracy of the method, expressed as E%, was thecloseness of agreement between the mean of a set ofanalytical results and the theoretical value. Satisfactoryaccuracy was achieved if E% was±15% (±20% near LOQ).

Carry-over

Memory effect, intended as carry-over of analytes fromsample to sample, was evaluated: two blank samples wereinjected after each run of spiked samples at 100 and 200 ng/mL. Two blank samples were also analyzed after realpositive samples.

Carry-over was considered negligible if the measuredconcentration was below the LOQ.

Stability

Samples stability in commercial buffer solutions and inpure OF, diluted with mobile phase and stored in theautosampler unit at 20 °C, was evaluated analyzing andquantifying spiked samples at 50 ng/mL for four consec-utive weeks. Samples stored at −20 °C were analyzed oncea week for 2 months, after freezing/thawing cycles.Acceptable stability was achieved if the sample could bequantified within a 20% error with respect to the expectedvalue.

Identification criteria

The criteria to be fulfilled for the identification ofanalytes were RT, the presence of three transitions andtheir relative ion intensities. RT should not vary morethan ±2.5%; relative ion intensities should not varymore than ±20% for ions with relative intensitiesof >50%, ±25% for ions with relative intensitiesbetween 10% and 50% and ±50% for ions with relativeintensities of <10%.

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in OF 613

Fig. 1 Transition chromatograms of a blank OF in DDS buffer and a blank OF in DDS buffer spiked with all the substances investigated at aconcentration of 20 ng/mL

614 S. Strano-Rossi et al.

Fig. 1 (continued)

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in OF 615

Tab

le2

Matrixeffect

ME%:area

oftheanalytein

matrix/area

oftheanalytein

mob

ileph

aseX

100(CV%)

Mavand

Cozart

Oralfluid

GBO

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

STD

ISTD

10(CV%)

50(CV%)

100(CV%)

10(CV%)

50(CV%)

100(CV%)

10(CV%)

50(CV%)

100(CV%)

10(CV%)

50(CV%)

100(CV%)

Amphetam

ine–

amphetam

ineD5

17(34)

18(29)

37(3)

36(3)

42(3)

40(4)

34(6)

37(7)

29(0.5)

28(1)

24(10)

24(11)

49(7)

51(2)

37(11)

39(11)

33(14)

34(14)

57(12)

74(9)

12(9)

13(11)

62(1)

65(2)

BEG–B

EG

D3

13(38)

15(39)

112(2)

111(1)

112(3)

109(1)

131(16)

124(13)

100(6)

99(4)

56(11)

54(11)

67(11)

73(10)

46(28)

53(27)

45(2)

48(2)

86(10)

79(9)

12(11)

13(9)

61(8)

66(8)

Cocaethylene–

cocaineD3

2(35)

12(30)

14(6)

14(6)

15(6)

15(5)

25(6)

18(4)

17(6)

12(1)

12(10)

17(9)

67(6)

38(3)

46(5)

32(4)

35(18)

28(12)

51(7)

32(0.5)

11(6)

12(6)

38(3)

26(4)

Cocaine–

cocaineD3

12(16)

12(30)

14(2)

14(6)

14(6)

15(5)

15(5)

18(4)

11(2)

12(1)

17(8)

17(9)

35(5)

38(3)

27(4)

32(4)

27(2)

29(3)

34(0.5)

32(0.5)

12(6)

12(6)

23(3)

26(4)

Codeine–

codeineD3

17(64)

18(44)

52(4)

53(2)

57(4)

57(4)

45(10)

54(7)

39(1)

41(1)

27(9)

28(11)

67(2)

73(4)

53(3)

62(4)

44(7)

47(8)

79(19)

78(12)

12(13)

12(9)

66(1)

70(3)

Ketam

ine–

ketamineD4

16(43)

16(40)

12(13)

14(12)

14(7)

15(9)

19(20)

24(12)

14(11)

17(10)

15(27)

16(27)

15(8)

15(0.5)

15(14)

14(22)

16(12)

16(28)

15(12)

16(20)

10(25)

12(14)

4(4)

4(17)

MDA–M

DA

D5

17(36)

19(40)

55(0.4)

54(3)

56(4)

55(3)

52(1)

61(2)

45(11)

44(10)

36(16)

36(13)

68(6)

77(6)

62(8)

68(4)

4716)

52(17)

108(9)

116(9)

13(13)

14(13)

91(6)

105(5)

MDEA–

MDEA

D5

16(41)

18(40)

56(1)

58(2)

56(5)

59(4)

59(6)

70(6)

50(5)

51(3)

31(17)

31(17)

51(2)

57(1)

40(20)

49(16)

34(18)

38(20)

65(11)

74(12)

12(10)

12(9)

54(5)

66(4)

MDMA–

MDMA

D5

16(34)

18(34)

53(1)

57(1)

57(2)

59(3)

60(2)

66(1)

44(5)

46(3)

31(14)

32(14)

59(1)

64(1)

47(5)

55(3)

40(8)

44(8)

70(14)

73(11)

12(10)

12(8)

62(2)

67(1)

Metam

phine–

metam

phineD5

17(36)

18(37)

58(2)

58(2)

66(1)

61(1)

60(1)

68(2)

51(1)

52(1)

33(13)

31(13)

58(5)

67(5)

50(3)

55(5)

45(2)

44(3)

54(11)

75(10)

12(8)

12(9)

64(2)

65(1)

Methadone–

methadone

D3

15(31)

16(40)

45(18)

45(18)

44(10)

41(14)

14(7)

15(3)

15(1)

15(4)

19(6)

18(4)

72(5)

78(3)

47(5)

52(4)

34(20)

34(22)

42(12)

40(12)

11(7)

11(9)

55(6)

58(6)

Morphine–

morphineD3

15(32)

18(38)

19(1)

23(1)

23(13)

27(10)

12(23)

21(29)

15(1)

18(7)

32(11)

38(10)

37(1)

63(5)

48(6)

66(4)

42(10)

54(6)

38(5)

57(7)

11(9)

12(22)

37(6)

52(5)

O-6-M

AM–O

-6-MAM

D3

14(54)

14(46)

34(2)

35(3)

37(1)

36(3)

60(5)

57(3)

41(5)

42(3)

26(17)

27(13)

82(1)

78(6)

58(5)

67(3)

49(6)

51(7)

92(15)

94(13)

2(12)

3(12)

80(3)

86(0.5)

THC–T

HCD3

26(58)

20(59)

48(4)

54(8)

41(8)

49(7)

10(64)

16(35)

15(5)

17(7)

14(7)

13(19)

149(9)

170(7)

53(8)

61(6)

27(18)

29(15)

126(18)

91(12)

12(21)

13(26)

56(4)

68(5)

Analyte—

deuterated

IS

616 S. Strano-Rossi et al.

Results and discussion

Method optimization

The main advantage of the proposed method is the directinjection of OF samples, without a preliminary samplepretreatment and the determination of all substances,including THC, in a single run of 8 plus 3 min of columnre-equilibration time. This allows the confirmatory analysisof the residual OF samples from on-site tests that aregenerally in very low amounts, with a unique analyticalmethod, thus requiring few microliters of sample.

The sensitivity achieved by the Jet Stream TripleQuadrupole Mass Spectrometer allows the analysis of thesamples without a preconcentration. As expected, the morecritical analyte was THC, which showed lower responsewith respect to other substances. The LC-MS/MS methodwas, therefore, optimized to achieve a satisfactory sensitiv-ity also for this substance.

Different mobile phases A and B were evaluated to reachoptimal analytical conditions. The best sensitivity for allanalytes was reached by adding formiate ammonium tomobile phase A and using acidified acetonitrile/methanolratio of 50:50 as phase B.

Further improvement for THC detection was representedby the optimization of MS/MS method, with the acquisitionof THC in an ad hoc window, optimized electromultiplier,capillary and nozzle voltages.

Validation results

The method developed allows obtaining the determinationof all analytes with satisfactory sensitivity, specificity and

repeatability. No significant interferences with the analytesby endogenous components of the OF or compounds fromthe devices were found. Analog substances and commonover the counter medicaments did not interfere in asignificant manner with the analytes of interest. Figure 1shows the transitions chromatogram of a blank OF (left)and of a blank OF spiked with all the substances at 20 ng/mL in DDS buffer (right).

Matrix effect studies, reported in Table 2, demonstratedthat the use of deuterated analogs of all substances balancedthe eventual ion suppression/enhancement effect of thematrix, as also reported by Concheiro [20]. As shown inTable 2, the matrix effect ME% (ion suppression/enhance-ment) was similar on the analyte and on its deuterated IS,apart from some exceptions at the lowest concentration.Therefore, deuterated analogs must be used to compensatefor matrix effect if an accurate quantification is needed.Anyway, even substances that show higher ion suppressioncan be detected with adequate sensitivity. Limits ofdetection and quantification achieved are reported inTable 3. Equations curves obtained in different matrices,with slopes and intercepts, correlation coefficients, and theirrelative standard deviations are reported in Table 4. Cali-bration curves obtained on drug/IS area ratios in mobilephase have statistically comparable slopes at a 5%significance level respect to those achieved with differentpreservative buffers plus OF and with neat OF; calculated Fvalues were always lower than the F critical, except forsome analytes (cocaine, BEG, MDEA, MDA, codeine, andTHC) in the GBO buffer, showing a matrix dependence forthese substances in GBO buffer.

Intra- and inter-day precision and accuracy results at low,medium and high levels for all the buffers and pure OF are

Analyte LOD (ng/mL) LOQ (ng/mL)

Rapidstat (DDS) Pure OF (GBO) Rapidstat (DDS) Pure OF (GBO)

Amphetamine 1 0.5 5 5

BEG 0.5 0.2 5 5

Cocaethylene 0.5 0.2 5 5

Cocaine 1 0.5 5 4

Codeine 1 0.5 10 5

Ketamine 0.5 0.4 2 2

MDA 1 1 5 2

MDEA <0.2 <0.2 5 4

MDMA <0.2 <0.2 4 2

Methadone 0.5 0.5 5 5

Methamphetamine 0.2 0.2 5 4

Morphine 5 4 10 5

O-6-MAM 2 1 4 2

THC 2 2 10 5

Table 3 Limits of detection(LODs) and LOQs obtainedwith different preservativebuffer of the collectiondevices

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in OF 617

Table 4 Linearity results in Mavand buffer (a), Cozart buffer (b), GBO buffer (c), oral fluid (d) and mobile phase A (e)

Analyte Intercept±SD (n=3) Slope±SD (n=3) r²±SD (n=3)

Amphetamine a −0.0195±0.0085 0.0076±0.0001 0.9998±0.0002

b −0.0148±0.0036 0.0074±0.0001 0.9999±0.0001

c −0.0346±0.0212 0.0067±0.00004 0.9992±0.0006

d −0.0050±0.0082 0.0070±0.00004 0.9993±0.0006

e −0.0033±0.0041 0.0072±0.0002 0.9997±0.0002

BEG a −0.0093±0.0017 0.0042±0.00001 0.9999±0.00003

b 0.0026±0.0020 0.0039±0.00003 0.9993±0.0003

c 0.0030±0.0096 0.0033±0.00002 0.9981±0.0018

d −0.0045±0.0005 0.0035±0.00004 0.9980±0.0017

e −0.0102±0.0093 0.0042±0.0001 0.9992±0.0014

Cocaethylene a −0.0843±0.0177 0.0121±0.0005 0.9987±0.0006

b 0.0191±0.0032 0.0053±0.0001 0.9993±0.0001

c −0.0042±0.0290 0.0040±0.0003 0.9985±0.0008

d 0.0204±0.0054 0.0038±0.0001 0.9983±0.0006

e −0.0561±0.1111 0.0110±0.0020 0.9948±0.0044

Cocaine a 0.0430±0.0438 0.0033±0.0001 0.9954±0.0034

b 0.0130±0.0009 0.0037±0.00004 0.9991±0.0008

c 0.0061±0.0028 0.0031±0.00002 0.9990±0.0004

d −0.0030±0.0005 0.0033±0.00003 0.9983±0.0015

e −0.0117±0.0066 0.0041±0.0001 0.9988±0.0019

Codeine a −0.0111±0.0124 0.0028±0.00001 0.9994±0.0002

b −0.0118±0.0015 0.0028±0.00004 0.9982±0.0028

c −0.0028±0.0045 0.0027±0.0001 0.9992±0.0005

d −0.0063±0.0028 0.0026±0.0001 0.9976±0.0016

e −0.0017±0.0057 0.0029±0.00003 0.9994±0.0004

Ketamine a 0.0145±0.0104 0.0046±0.0002 0.9978±0.0026

b −0.0378±0.0245 0.0053±0.0003 0.9939±0.0033

c −0.0125±0.0133 0.0059±0.00004 0.9983±0.0013

d 0.0100±0.0226 0.0058±0.0006 0.9995±0.0004

e 0.0119±0.0256 0.0048±0.0005 0.9995±0.0006

MDA a −0.02697±0.00543 0.01055±0.00018 0.99997±0.00001

b −0.0288±0.0086 0.0107±0.0002 0.9983±0.0028

c 0.0049±0.0095 0.0094±0.0002 0.9978±0.0035

d −0.0088±0.0039 0.0099±0.0001 0.9986±0.0018

e 0.0163±0.0127 0.0103±0.0001 0.9997±0.0002

MDEA a −0.0487±0.0073 0.0163±0.0001 0.9999±0.0001

b −0.0449±0.0038 0.0164±0.0002 0.9997±0.0002

c 0.0026±0.0095 0.0138±0.0004 0.9981±0.0017

d −0.0035±0.0069 0.0144±0.0001 0.9983±0.0020

e 0.0062±0.0052 0.0163±0.0003 0.9994±0.0006

MDMA a −0.01242±0.00323 0.00353±0.00004 0.9999±0.0000

b 0.0031±0.0108 0.0035±0.0001 0.9984±0.0027

c −0.0015±0.0105 0.0030±0.00003 0.9983±0.0010

d 0.0001±0.0012 0.0032±0.00005 0.9993±0.0006

e −0.0001±0.0015 0.0035±0.0001 0.9993±0.0011

Metamphetamine a −0.0238±0.0077 0.0112±0.0003 0.9997±0.0003

b −0.0197±0.0012 0.0111±0.0001 0.9995±0.0006

c −0.0436±0.0168 0.0101±0.0001 0.9987±0.0011

d −0.0168±0.0020 0.0106±0.00005 0.9983±0.0013

618 S. Strano-Rossi et al.

reported in Tables 5, 6, 7, and 8. Some results of repeatabilityand accuracy are sometimes out of the established ranges forcocaethylene probably due to the lack of its deuteratedanalog; therefore, we can only estimate its concentrations,using cocaine D3 as internal standard. Even codeine

concentration can only be estimated, as this substancesometimes is out of established ranges.

The method was linear in the range from the LOQ valueof each substance to 200 ng/mL, showing quadraticregression coefficients higher than 0.99 for all compounds.

Table 4 (continued)

Analyte Intercept±SD (n=3) Slope±SD (n=3) r²±SD (n=3)

e −0.0047±0.0080 0.0110±0.0003 0.9998±0.0003

Methadone a −0.2450±0.0626 0.0830±0.0017 0.9998±0.0001

b −0.5319±0.1836 0.0877±0.0028 0.9984±0.0013

c −0.0391±0.2107 0.0766±0.0023 0.9985±0.0014

d −0.0911±0.0449 0.0792±0.00003 0.9984±0.0014

e 0.1142±0.2624 0.0813±0.0017 0.9996±0.0004

Morphine a −0.0042±0.0087 0.0051±0.0001 0.9989±0.0006

b −0.0569±0.0478 0.0056±0.0002 0.9952±0.0035

c 0.0001±0.0315 0.0057±0.0004 0.9968±0.0040

d −0.0235±0.0076 0.0054±0.0001 0.9977±0.0031

e −0.0128±0.0452 0.0051±0.0004 0.9990±0.0007

O-6-MAM a −0.0128±0.0047 0.0027±0.00002 0.9995±0.0009

b −0.0087±0.0032 0.0026±0.00002 0.9984±0.0011

c −0.0052±0.0031 0.0024±0.00002 0.9991±0.0007

d −0.0042±0.0009 0.0026±0.00004 0.9986±0.0014

e −0.0025±0.0076 0.0028±0.0001 0.9994±0.0005

THC a −0.0058±0.0125 0.0039±0.0001 0.9995±0.0004

b −0.0271±0.0177 0.0032±0.0003 0.9985±0.0013

c −0.0269±0.0116 0.0032±0.0002 0.9992±0.0011

d −0.0147±0.0073 0.0035±0.0001 0.9990±0.0006

e −0.0270±0.0212 0.0039±0.0001 0.9987±0.0013

Table 5 DDS buffer repeatability (intra- (n=5) and inter-day (n=25) % variation coefficient of quantification) and accuracy (intra- and inter-day% error at 10, 50, and 100 ng/mL)

Substance CV% intra-day CV% inter-day E% intra-day E% inter-day

10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL

Amphetamine 5.96 4.75 3.16 6.34 5.09 3.31 15.90 −10.31 −7.10 16.63 12.11 2.20

BEG 1.25 0.86 1.17 4.45 1.14 1.24 4.47 −4.08 −2.91 6.53 4.36 3.17

Cocaethylene 17.01 15.10 9.32 18.38 2.31 3.38 15.77 12.11 9.87 21.17 18.55 21.54

Cocaine 1.19 1.75 0.85 15.52 1.75 1.41 2.02 −4.89 −3.98 0.45 1.36 0.08

Codeine 13.05 3.61 2.87 13.26 11.74 12.12 −15.44 8.11 14.45 6.40 3.55 10.71

Ketamine 11.52 8.25 7.24 12.22 3.0 2.03 3.31 2.69 1.25 2.07 6.34 1.23

MDA 1.98 2.83 3.12 2.27 3.14 4.42 −4.40 1.14 0.30 −7.68 6.51 2.19

MDEA 2.80 2.82 3.66 6.29 3.61 2.93 −9.26 6.91 −2.32 1.18 0.85 0.50

MDMA 2.65 0.86 0.79 4.43 1.61 1.76 1.35 −4.20 −1.71 5.64 3.14 2.42

Methadone 3.56 1.30 4.45 4.89 2.08 3.88 −2.72 0.66 −1.05 7.62 −3.62 −0.11Methamphetamine 4.23 2.03 2.60 5.24 2.85 2.39 1.90 −4.43 −2.12 6.96 3.23 0.67

Morphine 15.53 2.88 5.43 16.43 7.40 5.56 −7.81 4.67 5.72 18.49 9.11 7.40

O-6-MAM 5.86 2.06 4.68 16.11 12.17 7.60 2.69 15.03 9.09 18.23 12.36 8.63

THC 14.74 3.85 6.16 12.06 4.86 5.31 7.55 −4.15 −1.64 −7.06 −2.25 2.23

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in OF 619

No memory effect was observed, except for cocaine,after the injection of the 200 ng/mL spiked sample, at aconcentration below the LOQ, which appeared in the firstblank injection. It was observed a carry-over effect afterreal samples with high concentrations of cocaine (above1 μg/mL) that led to a quantifiable amount of cocaine(approximately 5 ng/mL) and BEG (below LOQ) in thefollowing injection.

Hence, after each positive cocaine sample, two injectionsof blank OF were performed. Samples showing drugsconcentrations above the upper level of the calibration curves

were diluted in 1:10 or 1:100, depending on the estimatedconcentration, with blank OF sample prior to a furtherinjection to achieve a correct quantification. Dilution integritywas demonstrated to be reliable, giving a E% of <15% ontriplicate samples at 1,000 and 10,000 ng/mL analyzed bydilution in 1:10 and 1:100 with blank oral fluid.

The analytes were stable in the samples for twomonths after eight freezing/thawing cycles, except formorphine and THC, that gave an error of >20%withrespect to the expected value after five and three cycles,respectively. The shelf life at room temperature was at

Table 6 Rapidstat buffer repeatability (intra- (n=5) and inter-day (n=25) % variation coefficient of quantification) and accuracy (intra- and inter-day % error at 10, 50, and 100 ng/mL)

Substance CV% intra-day CV% inter-day E% intra-day E% inter-day

10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL

Amphetamine 3.12 2.62 6.03 6.20 3.99 5.20 −1.76 3.84 −6.63 −7.95 −8.86 0.90

BEG 1.42 0.72 3.89 2.17 2.57 2.11 8.91 −5.72 −6.05 −8.03 −4.56 2.58

Cocaethylene 15.63 15.10 11.32 10.88 4.36 5.08 16.15 12.56 3.89 −4.11 13.12 5.84

Cocaine 3.42 3.09 1.38 10.53 14.88 8.59 12.24 −2.56 −9.20 17.31 8.15 −2.08Codeine 10.99 6.35 7.01 13.86 11.44 17.79 −15.74 11.04 −0.39 −0.68 −1.25 −12.41Ketamine 12.81 10.32 8.74 14.62 4.97 3.03 4.52 3.63 −1.15 3.87 8.12 2.73

MDA 1.98 4.43 2.86 4.00 5.71 2.43 8.39 −7.84 −7.08 13.11 3.19 −0.99MDEA 0.30 1.12 0.87 2.72 3.00 2.10 −2.72 −2.04 −2.26 17.71 8.85 0.07

MDMA 1.60 0.93 1.68 3.39 10.70 1.89 13.28 −2.45 −9.93 17.16 12.11 −11.03Methadone 5.16 3.75 4.51 3.89 5.99 4.89 3.79 −5.05 −2.48 7.52 3.25 −3.98Methamphetamine 3.36 1.50 4.68 4.90 9.52 3.93 10.97 −7.64 −5.23 16.04 −7.75 1.45

Morphine 15.97 7.17 2.38 17.44 12.67 14.44 5.43 −6.94 −4.32 4.76 −3.12 9.98

O-6-MAM 11.99 2.62 3.78 14.66 15.84 9.08 −15.30 11.97 1.88 14.44 5.86 3.18

THC 6.03 1.22 1.51 7.04 4.47 3.38 11.27 −11.99 −13.10 12.23 −12.25 −5.94

Table 7 Pure OF repeatability (intra- (n=5) and inter-day (n=25) % variation coefficient of quantification) and accuracy (intra- and inter-day %error at 10, 50 and 100 ng/mL)

Substance CV% intra-day CV% inter-day E% intra-day E% inter-day

10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL 10 ng/mL 50 ng/mL 100 ng/mL

Amphetamine 3.32 2.47 0.98 4.36 1.60 1.88 −12.02 −2.08 15.98 1.13 −2.12 9.18

BEG 3.03 4.23 3.05 4.57 4.42 2.43 −9.69 −9.12 3.43 1.33 −9.11 15.09

Cocaethylene 16.21 5.41 8.36 17.06 5.57 9.52 12.83 17.2 10.08 12.83 13.53 10.08

Cocaine 5.72 4.24 1.73 5.13 5.36 2.46 −10.47 −9.36 3.21 9.11 −9.42 11.66

Codeine 14.41 1.74 1.47 9.94 2.74 1.51 −4.85 −16.22 6.84 4.96 −16.21 4.66

Ketamine 7.71 8.15 4.56 10.72 3.90 2.11 3.22 4.11 2.15 1.46 6.72 2.11

MDA 5.46 0.97 3.89 4.89 2.14 3.39 −9.78 −6.05 11.16 4.35 −6.04 7.42

MDEA 3.22 2.63 3.8 4.32 2.82 2.91 −11.37 −2.54 3.68 3.27 −2.52 15.48

MDMA 8.28 2.48 5.75 8.28 2.34 4.12 −13.87 −6.27 11.91 −3.21 −6.31 8.82

Methadone 4.1 1.18 0.96 5.46 1.86 1.73 −15.7 −4.33 1.59 0.67 −4.32 7.66

Methamphetamine 6.61 2.25 1.60 5.64 1.87 1.96 −15.6 −6.6 4.51 1.60 −6.62 18.98

Morphine 9.83 7.09 6.78 10.89 5.35 3.93 −1.0 −17.77 16.2 −0.43 −15.0 9.77

O-6-MAM 10.87 3.76 2.48 5.89 2.37 3.41 1.83 −15.71 4.59 14.27 −15.71 4.64

THC 16.19 10.72 1.73 15.2 10.72 1.73 −15.3 −3.01 7.71 −15.3 −3.03 7.71

620 S. Strano-Rossi et al.

least 4 weeks for all spiked analytes, except morphineand THC, that gave an error of >20% after 2 and4 weeks, respectively, both in neat OF and in Rapidstatbuffer and that must therefore be considered unstable inthese matrixes for a storage of more than 2 weeks. Onthe contrary, both THC and morphine were stable in realsamples in DDS buffer and could be quantified withacceptable reproducibility and accuracy (CV% and E%of <20%) after all stability studies. Only in two spikedsamples, after two freezing/thawing cycles in neat OF, itwas observed the hydrolysis of O-6-MAM, cocaine, andcocaethylene with a simultaneous formation of morphineand BEG.

Method application

The method was applied to 466 real OF samplescollected during traffic controls. In 74 samples, cocainewas detected at concentrations ranging from under LOQto 13.5 μg/mL and BEG from 10 to 2 μg/mL. Forty-fiveof them contained also cocaethylene, in estimatedconcentrations ranging from below LOQ to 71 ng/mL.All subjects with cocaethylene had alcoholimetries above0.5 g/L, except for three of them.

THC was found in 49 samples, at concentrationsranging from amounts below LOQ to 1750 ng/mL.Ketamine was found in four samples, in concentrations

Table 8 GBO buffer repeatability (intra- (n=5) and inter-day (n=15) % variation coefficient of quantification) and accuracy (intra- and inter-day% error at 10 and 100 ng/mL)

Substance CV% intra-day CV% inter-day E% intra-day E% inter-day

10 ng/mL 100 ng/mL 10 ng/mL 100 ng/mL 10 ng/mL 100 ng/mL 10 ng/mL 100 ng/mL

Amphetamine 1.09 0.82 2.21 1.41 −1.36 −0.04 −2.11 −0.25BEG 1.06 0.41 1.88 0.87 0.43 −0.03 1.12 0.78

Cocaethylene 1.84 0.51 3.88 2.28 1.64 0.03 2.11 1.55

Cocaine 0.65 0.47 0.98 1.15 −0.96 −0.26 1.33 2.11

Codeine 0.43 0.87 1.22 2.23 0.28 −0.12 1.16 0.84

Ketamine 2.93 0.32 2.58 0.11 0.17 −0.15 2.1 1.99

MDA 2.89 0.50 3.51 1.21 −0.54 −0.23 0.11 0.96

MDEA 4.12 0.53 4.51 1.27 0.14 −0.11 0.55 0.76

MDMA 0.61 1.05 2.0 2.21 −1.55 −0.09 −2.21 −1.15Methadone 3.39 0.48 4.21 1.22 3.14 0.01 4.15 2.08

Methamphetamine 0.93 0.50 2.88 1.95 1.92 −0.04 0.51 0.98

Morphine 2.74 1.31 3.11 2.33 −3.37 −0.20 −5.58 −2.33O-6-MAM 1.69 1.36 2.84 2.37 −0.43 −0.07 −1.2 −0.88THC 1.77 0.92 2.15 1.55 −1.13 −0.13 −2.21 −0.55

Fig. 2 Real OF sample showing the presence of cocaine, BEG,cocaethylene, and MDMA (27, 97, about 10, and 106 ng/mL,

respectively)

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in OF 621

ranging from 17 to 94 ng/ml. MDMAwas detected in 11samples at concentrations between 2,616 and 327 ng/mL,with MDA and methamphetamine in four of them. Twosamples showed codeine and in one of them was alsopresent morphine.

Figure 2 shows a real OF sample in which were detectedcocaine, BEG, cocaethylene, and MDMA (27, 97, about 10,and 106 ng/mL, respectively). Figure 3 shows a THC-positive sample at 81 ng/mL.

As demonstrated by the results obtained, the methodallows the separation and identification of all substances ina single chromatographic run with good sensitivity and withvery short times of analysis, as sample pretreatment is notrequired and the UHPLC system allows very shortanalytical times. A further advantage is that the methodcan be directly applied also on OF collected with variousdifferent commercial devices. The four devices tested canbe considered indicative of the possible application of themethod to a wide range of different preservative solutions.

When compared to other published methods in scientificliterature, it exceeds others in its simplicity and speed ofanalysis. Previous papers published on OF analysis fordrugs of abuse generally perform a preliminary extractionof the samples [15–17, 21–24] also in case of LC/MSanalyses, or a protein precipitation and subsequent filtration[25]; other methods involve two chromatographic runs forthe detection of all the drugs [25]. A simple and effectiveprotein precipitation was performed by Concheiro et al.before LC/MS analysis [20], but the method does notevaluate THC, amphetamines and ketamine, substances ofprimary importance in DUID.

An issue for an eventual later gas chromatographic/MSconfirmation analysis can be the quantity of OF buffermixture available, which is sometimes too small, even afterdilution [26]. Anyway, it has been developed a GC/MSmethod that employed only 250 μL of OF and after various

extraction, derivatization steps and different GC/MS anal-yses, allowed the determination of all the analytes ofinterest [27].

The direct analysis of OF buffer mixture by UHPLC-MS/MS overcomes all these problems and analytical steps.In cases where available OF buffer mixture was less than250 μL, a proportional amount of internal standard mixturewas added and the sample could be directly analyzed.

Other studies compare results gained with OF to thoseobtained by plasma samples [28, 29] or both [25, 26,30–33] to determine the percentage of false-positive orfalse-negative results.

In the present study, the method was designed to allowthe confirmation analysis exactly on the same aliquot ofOF used for the screening, thus avoiding problemsrelated to different drugs concentrations in two differentsamplings.

The possibility to analyze directly the OF buffer mixturebypasses the issues related to extraction recoveries and alsosmall residual volumes of samples can be analyzed, evenlower than 250 μL. The results obtained from real samplesdemonstrate the suitability of the method for the confirma-tory analyses of samples from on-site testing.

Conclusions

The proposed method, fully validated, allows the analysisof several drugs of abuse in a single chromatographic runwith good sensitivity, linearity, repeatability and with veryshort time of analysis. A further advantage is that samplepreparation is not necessary and also OF sampled withcommercial kit, that dilute the specimen in unknowncomposition buffers, can be directly analyzed, thus, allow-ing the analysis on the same residual OF sample from thepreliminary screening test. This is possible because the

Fig. 3 Real THC-positive OF sample at 81 ng/mL: transitions of THC and THC-D3 (I.S.)

622 S. Strano-Rossi et al.

volume of OF needed for the analysis is very small, andthere is no need of a subsequent additional OF sampling.This method has been successfully applied on real OFsamples from on-site tests, showing a 16% of positivesamples for cocaine, 10.5% of positive for THC, 2.3% ofpositive samples for amphetamine-like designer drugs and a0.8% of ketamine-positive samples.

Acknowledgments The present work was supported by the Italian“Dipartimento delle Politiche Antidroga,” through the project“TOXTEST.” Special thanks go to the Italian “Dipartimento diPolizia Stradale” and to police officers that performed on-sitescreening tests and collected OF samples. We are also grateful toAgilent Technologies for its technical support.

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