Talanta 143 (2015) 50–55

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Talanta

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journal homepage: www.elsevier.com/locate/talanta

Rapid label-free determination of ketamine in whole blood usingsecondary ion mass spectrometry

Hua-Yang Liao a, Jung-Hsuan Chen b,c, Jing-Jong Shyue a,d, Chia-Tung Shun b,e,Huei-Wen Chen c, Su-Wei Liao e,1, Chih-Kang Hong e,1, Pai-Shan Chen b,e,n

a Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwanb Forensic and Clinical Toxicology Center, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei 100, Taiwanc Graduate Institute of Toxicology, National Taiwan University College of Medicine, National Taiwan University, Taipei 100, Taiwand Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwane Department and Graduate Institute of Forensic Medicine, National Taiwan University, Taipei 100, Taiwan

a r t i c l e i n f o

Article history:Received 19 January 2015Received in revised form23 April 2015Accepted 26 April 2015Available online 9 May 2015

Keywords:KetamineWhole bloodDrug screeningSecondary ion mass spectrometry (SIMS)Principal-components analysis (PCA)Gas chromatography–mass spectrometry(GC–MS)

x.doi.org/10.1016/j.talanta.2015.04.07440/& 2015 Elsevier B.V. All rights reserved.

esponding author at: Forensic and ClinicalUniversity College of Medicine and National00, Taiwan. Tel.: þ886 2 23123456x88879; faail address: paishanchen@ntu.edu.tw (P.-S. Chthors contributed equally to this study.

a b s t r a c t

A fast and accurate drug screening to identify the possible presence of a wide variety of pharmaceuticaland illicit drugs is increasingly requested in forensic and clinical toxicology. The current first-linescreening relies on immunoassays. They determine only certain common drugs of which antibodies arecommercially available. To address the issue, a rapid screening using secondary ion mass spectrometry(SIMS) has been developed. In the study, SIMS directly analyzed ketamine in whole blood without anypretreatment. While the untreated blood has a complicated composition, principal-components analysis(PCA) is used to detect unknown specimens by building up an analytical model from blank sampleswhich were spiked with ketamine at 100 ng mL�1, to simulate the presence of ketamine. Each char-acteristics m/z is normalized and scaled by multiplying the root square of intensity and square of cor-responding m/z, developed by National Institute of Standards and Technology (NIST). Using linearregression and the result of PCA, this study enables to correctly distinguish ketamine positive andnegative groups in an unknown set of specimens. The quantity of ketamine in an unknown set wasdetermined using gas chromatography–mass spectrometry (GC–MS) as the reference methodology.Instead limited by commercially available antibodies, SIMS detects target molecules straight despite thelabel-free detection capabilities of SIMS, additional data processing (here, PCA) can be used to fullyanalyse the produced data, which extends the range of analytes of interest on drug screening. Further-more, extremely low sample volume, 5 mL, is required owing to the high spatial resolution of SIMS. Inaddition, while the whole blood is analyzed within 3 min, the whole analysis has been shortened sig-nificantly and high throughput can be achieved.

& 2015 Elsevier B.V. All rights reserved.

1. Introduction

Fast screening for both legal and illegal drugs has historicallybeen accomplished using immunoassays (e.g. enzyme-linkedimmuno sorbent assay (ELISA) and enzyme multiplied immu-noassay technique (EMIT) [1–3]. They are commonly used as firstline screening methods in urine, blood or other bio-fluids [4–7].The screening is performed with rapid on-site devices based on animmunoenzymatic reaction. Although they are relatively fast andoffer high throughput analytical applications, immunoassays are

Toxicology Center, NationalTaiwan University Hospital,

x: þ886 2 23218438.en).

only able to analyze limited common medicinal drugs and drugs ofabuse where their antibodies are commercially available [8–10].Another main deficiency of immunoassays is the cross-reactivity.Compounds with structures similar to the target drug interferewith the results of immunoassays. The amounts of drugs cannot beaccurately measured, which leads to prevalence false positivereports or, more importantly, false negative screening results [11–13]. Therefore, a second analytical method, a chromatographicseparation (gas chromatography (GC) or liquid chromatography(LC), typically)–mass spectrometry (MS), has been developedeither as a complement to immunoassays in clinical testing or asthe analytical method in forensic and doping control applications[14–19].

A single MS experiment coupled with GC or LC is able to ana-lyze complex specimens, detect and characterize a large number ofknown compounds, as well as identify unknown substances. The

Fig. 1. Scaled spectrum for negative control and spiked sample group. The y-axis scaledby multiplying the root square of intensity and square of corresponding m/z toemphasize the high m/z region. The broken lines indicated the standard deviation.

Fig. 2. (a) Distribution of scores for each component (empty symbol) and its per-centage in variation (filled symbol). The standard deviation in scores is shown asbars. (b) PCA scores calculated using negative control (square) and spiked sample(circle) projected on PC1 and PC2 space. The elliptical confidence level is calculatedusing robust method. The scores of unknown samples calculated using linearregression are overlaid as triangles.

H.-Y. Liao et al. / Talanta 143 (2015) 50–55 51

techniques perform high sensitivity and provide well separation.However, majority specimens are subjected to different prepara-tion methods prior to analysis [20–24]. It is usually time con-suming and labor intensive. Important missions to be achieved areshortening pretreatment procedure and minimizing samplevolume required in both approaches. Alternatively, secondary ionmass spectrometry (SIMS), which sputters a specimen surface byion bombardment has been emerged to identify target analytes onthe biological samples. SIMS is capable of providing lateral reso-lution and imaging sensitivity on the order of parts-per-million[25–32]. SIMS is regarded as one of the most important micro-analytical tools in the semiconductor material industry for ana-lyzing trace amount of dopants and organic impurities. It allowsthe detection of the elements and small molecular fragments aswell as the separation of stable and radioactive isotopes on asurface of materials. Local concentration can also be measured byscanning the ion beam and collecting spectra pixel by pixel with alateral resolution commonly from 50 nm to 200 nm [29,33,34]. Tomap a complete surface of complex bio-sample, such as tissues orcells, it takes longer time to obtain enough counts in each pixel.For a homogeneous sample such as blood, an analysis focuses onextracting target analyte ions from a sample rather than profilingits surface, which means only few minutes are required to collectenough data.

With the high-energy ion bombardment, the molecularstructure in the surface tends to be broken hence SIMS typi-cally uses isotope or hetero-element labeling to aid the iden-tification of molecules of interest. With the recent develop-ment of cluster primary ion such as C60

þ , more surface loca-lized interaction and higher sputter rate are obtained and it ispossible to generate secondary molecular ions of high massthat identified molecular species directly [35–38]. In otherwords, label-free analysis of biological and organic surface canbe realized. To further enhance the intensity of these mole-cular ions and hence the sensitivity to molecular information,methods like enhanced oxygen-uptake [39], ion cosputtering[33,40], and optimization of analytical parameters [41] arealso being developed.

In the study, C60þ-based molecular SIMS has been developed to

determine the administration of ketamine in whole blood. Keta-mine, marketed as an anesthetic for human and veterinary, con-tinues to become popular in drug abuse scene. As its self-admin-istration behavior is similar to central nervous system (CNS)depressant drugs, ketamine abuse quickly spreads worldwide. Ithas been then replaced in the controlled substance in manycountries. Recent reports have indicated that ketamine abusersappeared to have severe bladder and kidney damage, such asulcerative cystitis, severe dysuria, or bladder dysfunction, whichare related to the effect of ketamine dose [42–47]. Biological fluidssuch as urine [48,49] and blood [48,50,51] are commonly used fordetermining the administration of ketamine. In Taiwan, a cutofflevel of 100 ng mL�1 is set in human urine. However, there is nocriteria for whole blood analysis. Whole blood usually has complexinterferences which cause many difficult analytical problems. Asample preparation is required before analysis. Therefore, it isimportant to develop a rapid and precise screening method. Totest the novel technique the same cutoff of ketamine is applied towhole blood analysis here. In the study, extremely low bloodvolume, 5 mL, was used for detection of ketamine without samplepre-treatment. GC–MS was employed as the reference methodol-ogy, for the qualitative confirmation as well as for the quantitativedeterminations of ketamine. Principal-components analysis (PCA)was performed on characteristic mass fragment intensitiesfrom each specimen. With the assistance of PCA, SIMS enabled tocharacterize the most chemically unique mass fragments of

ketamine in complex blood samples and remove much of theambiguity of similar low mass species that arose from biologicalsamples.

Fig. 3. PCA loadings at characteristic fragment of ketamine: (a) PC1, (b) PC2, (c) PC3 and (d) PC4. (m/z 125 and 127 [CH2ClC6H4]þ; m/z 152 and 154 [M–CO–NHCH3–C2H3]þ; m/z 165and 167 [M–CO–NHCH3–CH2]þ; m/z 179 and 181 [M–CO–NHCH3]þ; m/z 207 and 209 [M–NHCH3]þ; m/z 220 and 222 [M–OH]þ; m/z 224 and 226 [M–CH2þH]þ; m/z 238 and 240[MþH]þ; m/z 254 and 252 [MþCH3]þ; m/z 260 and 262 [MþNa]þ .)

Table 1Method validation for GC–MS confirmatory analysis.

Compound Linearity(ng mL�1)a

r2 LOD(ng mL�1)

Spiked blood

Concentration(ng mL�1)

RSD (%) intraday,n¼5

RSD (%) interday,n¼15

Calculated concentration(ng mL�1)

Difference (%)

Ketamine 20–20,000 0.9995 20 100 2.7 5.9 95.7 �4.310,000 3.1 5.0 9908.3 �0.9

a Blood sample spiked with 20, 40, 100, 200, 500, 1000, 5000, 10,000 and 20,000 ng mL�1, n¼3.

Table 2Ketamine analysis using SIMS and GC–MS.

Sample no. GC–MS SIMSConcentration (ng mL�1) Screening (100 ng mL�1)

105 ND �121 ND �144 ND �165 ND �186 404.2 þ194 109.4 þ199 263.5 þ322 ND �342 447.4 þ394 414.5 þ

ND: not detected.þ: ketamine positive.�: ketamine negative.

H.-Y. Liao et al. / Talanta 143 (2015) 50–5552

2. Materials and methods

2.1. Material and reagents

Ketamine standard solution (1 mg mL�1 in methanol), deuter-ated internal standard (IS), and ketamine-d4 (100 μg mL�1 inmethanol) were purchased from Cerilliant Corporation (Texas,USA). HPLC grade solvents, such as hexane and dichloromethane,were purchased from J. T. Baker (USA). Sodium carbonate (Na2CO3)was provided by Sigma-Aldrich (St. Louis, MO). Deionized waterwas purified in a Milli-Q RO Plus 60 filtration system (Millipore,Massachusetts, USA) before use for preparing aqueous solutions.

2.2. SIMS measurement

The instrument was based on a PHI 5000 VersaProbe scanningX-ray microprobe (ULVAC-PHI, Chigasaki, Japan) [48]. A Wien-fil-tered C60

þ ion source (IOG C60-10, Ionoptika, Chandler's Ford, UK)

H.-Y. Liao et al. / Talanta 143 (2015) 50–55 53

was operated at 10 nA (measured with an Au target) and 10 kV andrastered onto a 2 mm�2 mm area at the eucentric position withan incident angle of 70°. The quadrupole mass analyzer (EQS1000,Hiden, Warrington, UK) was located at 147° from the C60

þ ionsource with a 35° take-off angle and a working distance of 60 mm.The mass analyzer was optimized for a count rate of 12Cþ and them/z was calibrated with ionized C60 vapor at m/z 720. Duringacquisition, a flooding electron source biased at 1 V was used tocompensate for the charge-up effect. The sample surface is sput-ter-cleaned for 5 min to reach steady state; then 4–6 spectra wereacquired for each specimen. The typical sampling volume is in theorder of nL.

2.3. Multivariate analysis

Spectra acquired from negative control group and spikedsample group are analyzed by robust principal component analysis(robPCA) using ChemoSpec: Exploratory Chemometrics for Spec-troscopy (version 1.51-2) and SparseM: Sparse Linear Algebra(version 0.96) package in R: A Language and Environment forStatistical Computing (version 2.15.2) software. The intensity ateach m/z is first normalized by total intensity then scaled bymultiplying the root square of intensity and square of corre-sponding m/z, according to the mass spectral search softwaredeveloped by National Institute of Standards and Technology(NIST). For scaling the spectrum, because the ion yield decreasedsignificantly with increasing m/z, the information at higher m/zregionwhere the fragments of ketamine are present is emphasizedin order to down-play the contribution of elemental informationat low m/z. Unscaled covariance matrix is then used to digest thehigh-dimension spectra to principal components. For unknownsamples, spectra from each specimen is averaged, normalized andscaled; then linear regression is used to calculate the scores of first4 principal components.

2.4. GC–MS measurement

Analysis of ketamine was performed on a Thermo Trace GCUltra gas chromatograph instrument (Texas, USA) equipped with asplit/splitless injector and coupled with a thermo ISQ mass spec-trometer (Texas, USA). A 30 m DB-5MS fused silica capillary col-umn (0.25 mm I.D., 0.25 mm film thickness) was used for separa-tion of the analyte. Initially the column temperature was at 130 °Cfor 1 min, and then it was ramped to 170 °C at 25 °C min�1. It wasfurther raised to 200 °C at 20 °C min�1 and kept for 4 min; it wasthen increased to 280 °C at 60 °C min�1 and held for 2 min. Thepurity of helium carrier gas was 99.9995%, and the flow rate was1.0 mL min�1. The inlet was operated at 300 °C and was used inthe pulsed splitless mode. Ionization was operated in the electronimpact (EI) mode at 70 eV. The temperature of the ion source was230 °C and the temperature of the quadrupole mass filter was150 °C. The mass spectrometer (MS) was operated in the total ionchromatogram (TIC) mode and a mass range of m/z 50–250 wasscanned to confirm the retention time of ketamine. The selectedion monitoring (SIM) mode was used for the determination of thetarget compound. Three ion transitions for ketamine were mon-itored as follows: 180 (quantification), m/z 182 and 209.

2.5. Standard and working solutions

Ketamine was prepared in 10 mL of methanol to obtain astandard stock solution with a concentration of 100 mg mL�1,which was stored at �20 °C. A working solution of 10 mg mL�1

containing ketamine was prepared in methanol every week andstored at 4 °C. A series of working solutions were made by dilutingthe 10 mg mL�1 solution with methanol daily and stored at 4 °C.

2.6. Sample screening using SIMS

The samples were human whole blood from forensic cases. Thesamples were vortexed for 2 min. 5 μL of blood was spotted on apiece of 52 nm thick thermal oxide on Si wafer (1 cm�1 cm). Thewet blood spots were then placed under a nitrogen flow to drynessfor 30 min. During the initial evaluation of SIMS, a portion of 1 mLblank whole blood was spiked with 10 mL, 10 mg mL�1 of ketamine,which was considered as spiked sample group and followed theabove procedure.

2.7. Sample preparation for GC–MS analysis

To 0.5 mL of each whole blood sample, 20 mL of 10 mg mL�1

ketamine-d4 (internal standard, IS) was spiked. 1 mL of 0.1 MNa2CO3 (aq) and 1.5 mL of dichloromethane: hexane (1:3 v/v) wereadded to blood samples, which were then vortexed for 2 min. Themixture was centrifuged at 4000 rpm for 10 min. 500 μL of theorganic phase was transferred into a 12 mm�75 mm glass tubeand evaporated to dryness under nitrogen at 40 °C (approximately8 min). The residue was reconstituted in 50 mL of ethyl acetate and1.5 mL was injected into the GC–MS for further analysis.

2.8. Confirmatory analysis by GC–MS

The samples found containing no measurable amount of keta-mine using the GC–MS method were used as negative control andfor the preparation of calibration curve. The concentrations usedfor calibrants were 20, 40, 100, 200, 500, 1000, 5000, 10,000 and20,000 ng mL�1. Two controls spiked ketamine at 100 and10,000 ng mL�1 were prepared in whole blood described above.

For identification, ion ratios were calculated for ketamine in thesamples using the ions m/z 180, 182, and 209, and compared withthose from a QC containing 100 ng mL�1 of ketamine. The con-centration of ketamine in each sample was interpolated from thecalibration line constructed using a ratio of the peak area ofketamine (m/z 180) to that of ketamine-d4 (m/z 184). Linearregression lines were calculated and the correlation coefficientswere better than 0.995 in all cases.

Positive results were reported if the concentration of ketaminewas greater than 100 ng mL�1. The criteria applied for acceptableGC–MS identification were those used routinely in our laboratory.The relative retention time of the substance to be identified shouldnot differ by more than 2%. Moreover, the abundances of at leastthree diagnostic ions should not differ by more than 20% relativefor ion abundances. The abundance of a diagnostic ion must bedetermined from integrated ion chromatograms, where theabundance of the least intense ion must have the signal to noiseratio greater than 3:1.

3. Results and discussion

The aim of this study was to screen ketamine samples in wholeblood applying no sample preparation. Our approach used GC–MSto confirm the concentration of ketamine in all specimens. A groupdetermined without the presence of ketamine was considered asblank and negative control group. Though SIMS does not allow theseparation of targets and interferences, the use of instrument withautomated fragmentation with PCA model enables the distinctionsto be made. To optimize the parameters of SIMS and PCA model,blank whole blood was spiked with ketamine at 100 ng mL�1.

The scaled spectra for ketamine spiked sample and negativecontrol are shown in Fig. 1. Owing to the complex mixture of thewhole blood, the pattern of ketamine was overlapped by strongbackgrounds and it was not possible to distinguish the

H.-Y. Liao et al. / Talanta 143 (2015) 50–5554

administration of ketamine. Using PCA, the screening plot (Fig. 2a)indicated that the first 4 PCs described more than 90% of varia-tions. Hence 4 PCs were used for subsequent analysis. It is clearthat while the PC1 described the most (59%) variation, the scorecannot completely discriminate the spiked samples from negativecontrols. On the other hand, the PC2 clearly separated the differentgroups while described 23% of variance.

By plotting the scores in PC1 and PC2 space, Fig. 2b revealsclear discrimination of sample groups where spiked samples arelocated at upper-left quadrant and negative controls are at lower-right quadrant. In other words, negative PC1 and positive PC2suggest the presence of ketamine abuse. For easier comparison,the referencing spectrum of ketamine was scaled and overlaid onthe loading and the loadings of characteristic ketamine fragmentsare listed in Fig. 3.

It is clear that besides [MþH]þ of ketamine (with 37Cl at m/z240), [M–CH2þH]þ (at m/z 224 and with 37Cl at 226),[M–NHCH3]þ (with m/z 37Cl at 209) and peaks at lower m/z thatmay overlap with other species, negative PC1 loadings corre-sponding to the characteristic molecular fragments of ketamineare found. Therefore, lower PC1 score may suggest the presence ofketamine. However, owing to the overlapping of PC1 scores pre-vent a clear discrimination of spiked samples from negative con-trols (Fig. 2a), PC1 alone cannot be used to identify the adminis-tration of ketamine.

For PC2, positive loadings corresponding to [MþNa]þ ,[MþCH3]þ , [MþH]þ , [M–CH2þH]þ , [M–NHCH3]þ , [M–CO–NH-CH3]þ , [M–CO–NHCH3–CH2]þ , [M–CO–NHCH3–C2H3]þ and [CH2-ClC6H4]þ (with 37Cl at m/z 240) were identified. Therefore, thepositive PC2 clearly indicated the presence of ketamine. While thehigher background at high mass shows negative loading of PC1and positive loading of PC2, it may be argued that the backgrounddominated the separation of groups. However, since the averagedspectra showed comparable background (Fig. 2) and the analysis ofvariance (ANOVA) reveals no significant difference between thesample groups at the level of 0.05, the effect of the backgroundwas dismissed.

Using the first 4 PCA loading determined with spiked sampleand negative control, scores of spectra obtained from unknownsamples were calculated using linear regression. The resultingscores (triangle) are overlay on the PC1 and PC2 space as shown inFig. 2b. The analysis suggests that ketamine was present over the100 ng mL�1 in samples 186, 194, 199, 342, and 394 while samples105, 121, 144, 165 and 322 were negative. To validate the result, thepresences of ketamine in these unknown samples were analyzedusing GC–MS method.

For confirmatory analysis, liquid–liquid extraction was appliedprior to GC–MS analysis, not least to pre-concentrate ketaminefrom whole blood. The SIM mode also eliminated the contributionof interferences. Blank samples were spiked with ketamine formethod validation. The estimated linearity is shown in Table 1. Acalibration curve was constructed for different concentrationsranging from 20 to 20,000 ng mL�1 in whole blood. The coefficientof determination (r2) was 0.9995. The limit of detection (LOD) was20 ng mL�1. Intraday relative standard deviations (RSDs) were2.7% and 3.1% at concentrations of 100 and 10,000 ng mL�1,respectively. Interday RSDs were 5.9% and 5.0% at concentrationsof 100 and 10,000 ng mL�1, respectively.

Three diagnostic ions were used to identify ketamine in GC–MS(m/z 180, 182 and 209) corresponded to those observed in SIMS.Five of 10 unknown samples were confirmed the administration ofketamine, which were samples 186, 194, 199, 342, and 394. Keta-mine was not detected in the other samples. Comparison of SIMSwith the more established GC–MS showed good agreementbetween the methods (Table 2). It is worth noting that in Fig. 2bthe negative unknown cases are located far from the overlay area

with the positive group. On the other hand, the positive samples194 and 199, which confirmed relative lower ketamine con-centrations (109.4 and 263.5 ng mL�1, respectively) are close tothe border of the two groups. The other positive samples whichcontained ketamine more than 404 ng mL�1 spread away from theoverlay area. This result indicates that the SIMS–PCA method iscorrelative to the regular GC–MS methods and could be used forrapid screening with extremely low sample consumption.

4. Conclusions

The proposed label-free SIMS–PCA method offers a promisingalternative to the conventional drug screening methods. Thecommon immunoassay cannot provide an accurate measurementnear cut-off values and generate false reports. Screening usingSIMS with PCA has been developed a complement to immu-noassays in clinical and forensic testing. It successfully dis-tinguished the difference of positive and negative ketamine groupsfrom complex blood samples. Although SIMS with PCA is not reallysuitable for quantification, it provides a quick pre-screening priorto routine GC–MS analysis. It requires extremely low volume (5 mLin this work and the actual analysis volume is mm�mm�nmhence only pL is required in principle) of whole blood and nosample preparation is necessary. In other words, it is ideal for traceanalysis. In addition, SIMS is straight forward to automate the dataacquisition and it only takes a few minutes for each sample and issuitable for rapid, high throughput analysis. The study provides adesirable method on drug screening in terms of accuracy, speedand sample volume.

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