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Journal of Chromatography A, 1277 (2013) 1– 6

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

jou rn al h om epage: www.elsev ier .com/ locat e/chroma

etection of ketamine and its metabolites in human hair using an integratedanoflow liquid chromatography column and electrospray emitter fritted with aingle porous 10 �m bead

ark C. Parkina,∗, Alana M. Longmoorea, Sophie C. Turfusa,1, Robin A. Braithwaitea,avid A. Cowana, Simon Elliottb, Andrew T. Kicmana

Analytical and Environmental Sciences, King’s College London, 150 Stamford Street, London SE1 9NH, UK(ROAR) Forensics Ltd, Malvern Hills Science Park, Geraldine Road, Malvern, Worcestershire WR14 3SZ, UK

r t i c l e i n f o

rticle history:eceived 29 August 2012eceived in revised form1 November 2012ccepted 11 December 2012vailable online 19 December 2012

eywords:ano-electrosprayanoflow liquid chromatographyair analysis

a b s t r a c t

Targeting metabolites incorporated into hair following drug administration is useful for evidential pur-poses as this approach can aid in differentiating between administration and passive exposure. Greateranalytical sensitivity is required than for targeting the parent drug alone. A 20 �m i.d. fused silica capil-lary column with an integrated electrospray emitter fritted with a single porous 10 �m polymeric beadhas been successfully fabricated to facilitate this purpose. The sensitivity gains through the use of theseintegrated single fritted columns coupled to a nanoelectrospray source (nanoflow–LC nanoESI) over con-ventional liquid chromatography–tandem mass spectrometry (LC–MS/MS) columns was explored bytheir application to the detection of ketamine and its phase I metabolites in human hair. Hair was collectedfrom 4 volunteers following the administration of a small oral dose of ketamine (50 mg) and subsequentlyanalysed by the capillary–LC nanoESI approach. The drug and its metabolites were extracted from hair

etamine using solid phase extraction following a methanolic wash, pulverisation with a ball mill and acid diges-tion. From a 50 �L extract, 1 �L was injected and the method provided a limit of detection estimated tobe 5 fg on column for ketamine and norketamine and 10 fg for dehydronorketamine. The single porousfrit minimises extra column band broadening and offers an alternative fritting approach which reducesthe blocking of the electrospray emitter, in comparison with other approaches such as sintering andpolymerisation.

. Introduction

The use of samples of hair, saliva and sweat are increasinglyecoming attractive alternative matrices to conventional speci-ens of blood and urine for the detection of drug administration

n forensic toxicology [1–3]. Many drugs are rapidly excreted fromhe body and the analysis of blood and urine is only suitable foretecting recent administration, typically for a few days. Follow-

ng administration, drugs can be trapped into the shaft of eachair growing out of its follicle. As the hair grows over weeks toonths, a “time-capsule” of drug use is created with the drug band

n the hair closest to the scalp showing most recent use. The win-ow of detection in hair is usually only limited by its length andan range from a few weeks to years. Changes in the concentration

∗ Corresponding author. Tel.: +44 20 7848 4879; fax: +44 20 7848 4980.E-mail address: mark.parkin@kcl.ac.uk (M.C. Parkin).

1 Current address: Victorian Institute of Forensic Medicine, Southbank, Victoria,ustralia.

021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2012.12.019

© 2012 Elsevier B.V. All rights reserved.

of incorporated drug concentrations can be brought about bythe cosmetic treatment of hair such as bleaching and dyeing[4,5].

The exact mechanisms of drug incorporation into hair from thebloodstream are not well understood, but it is known that bindingwith the pigment melanin and also the protein keratin are majorfactors for basic compounds [6–10]. The amount of drug incorpo-rated in hair is usually very small, ranging from 0.01 to 5000 pg/mgof hair for frequently abused and therapeutic drugs [11]. GC–MSand more recently LC–MS/MS [12] is being applied to achieve theanalytical sensitivity and specificity required for forensic toxico-logy. Even so, to advance the field of hair analysis, greater sensitivityis highly desirable to facilitate the detection of such small concen-trations of drugs and their diagnostic metabolites. The targetingof drug metabolites in hair can aid in differentiating betweenadministration and passive exposure, a particularly important con-

sideration in environments where insufflation of a drug can causecontamination, e.g. cocaine, amphetamine and ketamine.

The hyphenation of LC to MS using atmospheric pressure ioni-sation sources has also been accompanied with the use of columns

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ith smaller internal diameters. ‘Narrow bore’ or ‘micro bore’olumns operate with flow rates more suitable for electrospray ion-sation, usually around 200–500 �L/min [13]. Recently, so callednanoflow” columns made from fused silica, having diameters100 �m and typically operating at nL/min flow rates have beenntroduced and used mainly in protein and peptide bioanalysis14]. By injecting the same amount of analyte and reducing theolumn i.d., analytes can be concentrated on column by large fac-ors. This trend towards column miniaturisation leads to a lowerilution of analytes in the column due to the lower flow ratessed and results in increased sensitivity of detection [15,16]. How-ver, smaller injection volumes, typically 50–100 nL are requiredn order to avoid zone dispersion and overload, compromising col-mn efficiency and sensitivity. This can offset the advantages ofreater concentration but can be circumvented through using “on-olumn focusing” where the analytes are concentrated onto theop of the column by injecting the analytes in a non-eluting solvent17].

A further direct consequence of the lower flow rates in nanoflowC is the ability for direct coupling to a miniaturised variantf electrospray ionisation known as nanoelectrospray (nanoESI).anoESI utilises low flow rates through the inlet capillary and

ensitivity gains are due both to the increased sample concentra-ion of the nanoflow LC and increased ionisation efficiency [18,19].olumns suitable for nanoESI–MS can be manufactured using fusedilica tapered to a narrow end producing an integrated ESI emit-er [20]. Integrated emitters reduce post-column dead volumehat can lead to band broadening and increase analytical sensi-ivity by improving chromatographic resolution. A challenge inhe construction of columns with integrated ESI emitters is thehoice of frit that is used to retain the functionalised silica beadshat are packed into the capillary. Methods that have been pro-osed include sintering [21], sol–gels [22] and a polymer monolith20].

This combination of capillary size LC together with nanoelec-rospray sources has been predominantly used by researchersnvestigating proteins and peptides by mass spectrometry and isather underexplored for its use in small molecule analysis espe-ially for drugs of abuse in alternative matrices. In this work, weemonstrate a process for incorporating an integrated electrospraymitter fritted with a 10 �m porous bead affording a sensitiv-ty that is useful for evidential purposes. This is similar to theritting approach demonstrated by Zhang et al. for capillary elec-rochromatography [23] but on a 5-fold smaller scale and with anntegrated electrospray emitter. To evaluate its capability, modelompounds targeted were ketamine and its metabolites in hairamples collected from volunteers following a small oral dose ofetamine hydrochloride.

. Experimental

.1. Chemicals and reagents

Ketamine–HCl was from Sigma Aldrich (Poole, UK). Stockethanolic certified standard solutions of norketamine–HCl

1 mg/mL as the free base) and dehydronorketamine (0.1 mg/mLs the free base) were from LGC Promochem (Teddington, UK).igh purity methanol, acetone, dichloromethane, acetonitrile,ropan-2-ol, ammonia solution, glacial acetic acid, formic acid,ydrochloric acid, hydrofluoric acid, sodium hydroxide, calcium

hloride and sodium acetate trihydrate were from Fisher Scien-ific (Loughborough, UK). All chemicals and reagents were of highurity analytical or HPLC grade. Water was ultra-pure filtered toPLC purity (resistivity 18.2 M� cm) from an Elga Purelab Maximaystem (Marlow, UK).

gr. A 1277 (2013) 1– 6

2.2. Drug extraction from hair samples

Hair samples were obtained from healthy, non-drug using vol-unteers as part of a drug administration study. Approval was givenby the Research Ethics Committee of King’s College London andinformed written consent was obtained in accordance with ourinstitutional procedures. Hair was collected from volunteers whohad been administered a small (50 mg) oral dose of a pharmaceu-tical preparation of ketamine (Ketalar®, Pfizer) with water. Thespecimens of hair were taken 2–3 months following the admin-istration. A collection of hair strands, 5 mm thick, was cut from thevertex posterior using scissors and cutting close as to the scalp aspossible. Hair was stored in aluminium foil at room temperatureuntil ready for analysis. Five, 5 mm long sections of hair were cutfrom the scalp end of the 5 mm thick sample for each volunteer andlabelled A–E. Each section of hair was weighed and then washed bysonicating it in 3 mL of methanol in a sonicating water bath for1 h. The methanol was decanted and another 3 mL of methanol wasadded and then decanted and combined with the first fraction. Thecombined methanol wash was evaporated under nitrogen at 60 ◦C.The dried wash was then reconstituted in 50 �L of 0.1% formic acidand vortex mixed for 30 s before being transferred to a chromato-graphic vial and analysed for the presence of any analytes. The hairwas then allowed to air dry at room temperature overnight. Driedhair sections were pulverised for 4 min at 45 oscillations per sec-ond with a Fritsch P23 Mini Mill Pulverisette tempered steel ballmill and ball bearings (C. Gerhardt UK Ltd., UK). The ball mill wascleaned between each segment pulverisation by rinsing out the millthree times with a solution methanol/water (50:50 v/v). Pulverisedhair samples were incubated with 1 mL of 0.1 M HCl overnight at55 ◦C. Following incubation, the hair was vortexed for 30 s and thencentrifuged at 5000 × g for 5 min and the supernatant was decantedand transferred to a clean tube. A second 1 mL aliquot of HCl wasadded to the hair and the procedure of vortexing and centrifugationwas repeated for a second time. The HCl fractions were pooled andneutralised with 2 mL of 0.1 M NaOH. The sample was then adjustedto a pH of 5.5 by the addition of 1.5 mL of 10 mM sodium acetatebuffer (pH 5.5). Ketamine and its metabolites were extracted fromthe buffered sample by solid-phase extraction (SPE). Varian Bond-Elut Certify I mixed-mode (cation exchange and C8) cartridges(Agilent, UK) were preconditioned by slowly passing under slightvacuum 3 mL of methanol followed by 1 mL of water and 1 mL of2 M glacial acetic acid. Buffered sample was then loaded onto thecartridge and allowed to slowly pass through the cartridge withoutvacuum. The cartridge was rinsed sequentially with 3 mL of water,1 mL of 0.1 M HCL and 3 mL of methanol slowly under vacuum.Retained drug and metabolites were eluted under gravity with 3 mL(added 1 mL at a time) dichloromethane/propan-2-ol/ammoniumhydroxide (78:20:2 v/v). The resulting eluent was evaporated undernitrogen at 60 ◦C. The dried extracts were reconstituted in 50 �L of0.1% formic acid and vortex mixed for 30 s before being transferredto a chromatographic vial. Drug-free samples of hair were obtainedfrom the volunteers on the day of the drug administration and theseblank samples were subjected to the extraction process and wereanalysed between post-administration volunteer samples.

2.3. Preparation of nanoflow–LC columns with integratednanoelectrospray emitters fritted with a single 10 �m bead

Fused silica capillary (360 �m o.d., 20 �m i.d.) was obtainedfrom Polymicro Technologies (Phoenix, AZ, USA). Highly porous(4000 A) 10 �m polymeric beads (sold as Varian PLRP-S media)

were kindly donated by Varian (Agilent Technologies, Edinburgh,UK). To prepare the single porous fritted nanoflow–LC columnswith integrated nanoelectrospray emitters, the fused silica capil-lary was cut to a length of 20 cm using a diamond tipped scribe.

M.C. Parkin et al. / J. Chromatogr. A 1277 (2013) 1– 6 3

Fig. 1. Fritting the 20 �m i.d. nanoflow column with a single 10 �m porous bead. (A) The distal end of the fused silica capillary is lightly tapped downward onto the monolayerof beads on a glass slide. (B) A single bead can be drawn into the capillary by wiping a tissue dampened with acetone towards the distal end. (C) The single bead is pushedt ne thrs

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owards the internal taper of the integrated electrospray emitter by flushing acetotationary phase packed behind.

1 cm portion of the polyimide outer coating was removedrom the capillary 5 cm from one end by burning using the flamerom a micro blow torch. A P-2000 laser puller from Sutternstruments (Intracel, Hertfordshire, UK) was used to draw thentegrated nanoelectrospray emitter from the capillary by placinghe polyimide-free portion into the laser path. The P-2000 was setith a programme with the following parameters (unitless); heat

50, filament 0, velocity 30, delay 128 and pull 125. The resultingip was etched back to an i.d. of ∼3 �m with 50% hydrofluoric acidor 30 s (use with caution and neutralise with CaCl2) [20,24]. Aingle 10 �m polymeric bead was placed at the head of the inte-rated column and emitter, where the pulled nanoelectrosprayip internally tapers down to 10 �m (Fig. 1). This was achieveds follows: a 10 �L aliquot of a 1 mg suspension of polymericeads in 1 mL of acetone was spotted onto a glass microscopelide. The acetone was left to evaporate leaving a monolayer ofeads on the slide surface. The distal end of the column was lightlyapped onto the centre of the spot of beads. This was sufficiento introduce a single bead into the end of the column and othereads that were on the outside of the capillary were wiped awaysing a piece of dry tissue. At this stage the capillary was examinednder a microscope to visualise that a single bead was in place

nd that further beads had been cleaned away from the outside.f multiple beads have entered the column, the end is cut off usinghe diamond scribe and the process repeated. A tissue dampenedith acetone was then wiped across the distal end of the column,

ough the capillary by an attached syringe. (D) The complete frit with the 3 �m C18

capillary action drawing acetone into the column sufficient tomove the single bead further into the column. The fritting processis illustrated in Fig. 2. An Upchurch Scientific Luer-to-MicrotightAdapter (Presearch Limited, UK) was used to connect an acetone-filled syringe to the distal end of the column. By applying fingerpressure to the syringe the acetone pushed the bead up to theinternal taper of the nanoelectrospray emitter to frit the column.The column was then slurry-packed with Varian 3 �m MicrosorbC18 reversed-phase particles (Agilent, UK), at 1500 psi to a lengthof 10 cm using nitrogen through a Nanobaume high pressurepacking bomb (Western Fluids Engineering, USA). The column wasattached to the exit of the bomb and the slurry was placed in a1 mL glass vial inside and was constantly stirred using a magneticstirrer. A nitrogen cylinder was attached to the bomb inlet andwas adjusted to deliver gas flow through the bomb at 1500 psi.

2.4. Tandem mass spectrometry with nanoelectrospray ionisation

Selected reaction monitoring (SRM) experiments were per-formed with nanoelectrospray ionisation using an Agilent 6460triple quadrupole mass spectrometer normally used with anAgilent HPLC-Chip Cube interface. In order to accommodate our

in-house integrated nanoelectrospray emitters, the Agilent ChipCube Interface Assembly (part number G1982-60050) was usedtogether with the Agilent Electrode Holder Assay (part numberG1982-60051) in order to guide and position the integrated column

4 M.C. Parkin et al. / J. Chromato

Fig. 2. An illustration of the fritting process. (A) First, the column is tapped down-wards onto the monolayer of 10 �m beads. (B) Usually, a single bead will enter thedistal end of the column and excess beads on the outside are wiped away using a drytt(i

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of a 1 �L sample loop did not cause overloading of the nanoflowcolumn.

Limits of detection (LOD) for ketamine, norketamine and dehy-dronorketamine using aqueous standard solutions were evaluated.

issue. (C) A corner of a new piece of tissue is damped with acetone and placed athe distal end of the column. Acetone is drawn into the column by capillary action.D) The plug of acetone continues to draw the bead further into the column wheret can be subsequently pushed to the taper using a syringe filled with more acetone.

nd emitter in front of the entrance to the mass spectrometer. Theanoelectrospray source conditions were optimised in positive

on mode as follows: capillary voltage 1900 V; source temperature00 ◦C. Following optimisation for each analyte, the following tran-itions, collision energies (CE) and fragmentor potentials (FP) weresed: m/z 238 → m/z 125 (CE 50 V, FP 120 V), m/z 238 → m/z 179 (CE5 V, FP 100 V), m/z 238 → m/z 207 (CE 10 V, FP 50 V) for ketamine;/z 224 → m/z 125 (CE 20 V, FP 160 V), m/z 224 → m/z 179 (CE 10 V,

P 160 V), m/z 224 → m/z 207 (CE 8 V, FP 150 V) for norketamine;/z 222 → m/z 142 (CE 15 V, FP 180 V), m/z 222 → m/z 177 (CE 15 V,

P 180 V), m/z 222 → m/z 205 (CE 6 V, FP 140 V) for dehydronorke-amine. The dwell time was set at 100 ms for each analyte.

.5. Nanoflow liquid chromatography

The distal end of column was connected to one port of an Agilent-port micro switching valve. The nanoelectrospray voltage wasrounded at this liquid junction using a crocodile clip and connect-ng lead from the valve port to the side of the Chip Cube Interfacessembly. A 20 �m i.d. fused silica capillary from Polymicro Tech-ologies was connected from the outlet of an Agilent 1200 nanoflowump with a built-in degasser to the 6-port valve. Sample injec-ions of 1 �L of the extract were performed manually using a 1 �Loop attached to the 6-port valve. Mobile phase solvents consistedf 0.1% formic acid for A and 0.1% formic acid in acetonitrile for B.air extracts were focused on-column by flushing to the head of

he column at 150 nL/min using 100% solvent A for 8 min follow-ng loading and injection. To begin separation, a 20 min gradientlution of 10%–80% solvent B at a flow rate of 150 nL/min was per-ormed followed by a 5 min equilibration period with 100% solvent

at 150 nL/min.

. Results and discussion

.1. Formation of a single particle 10 �m frit

Fig. 1 shows the 10 �m frit in a 20 �m i.d. fused silica capillaryolumn with an integrated nanoelectrospray emitter. Compared to

ther fritting methods such as in situ photopolymerisation [20] orintering glass particles [21], the frits were readily placed at theoint of tapering of the nanoelectrospary emitter. The single beadrit is retained by frictional forces and prevented from being forced

gr. A 1277 (2013) 1– 6

out of the column by the internal taper of the laser-pulled electro-spray emitter. This process served to minimise extra column bandbroadening and gave rise to reproducibly sharp peaks for a standardsolution of ketamine (100 pg/mL), with a peak width at half heightof 7 ± 1 s (n = 4, CV = 11.6%) for each column. A further benefit of frit-ting nanoflow columns this way was the elimination of the problemassociated with unsintered particles or damaged polymer breakingaway from the frit and blocking the electrospray emitter.

3.2. Nanoflow liquid chromatography tandem mass spectrometryof ketamine and its phase I metabolites

Four columns were prepared in order to determine the repro-ducibility of packing length and the retention behaviour betweencolumns for the first eluting (dehydronorketamine) and last eluting(ketamine) compounds. The mean length (n = 4) was 10.15 cm witha CV of 5.4%. The mean retention time for ketamine was 14.5 minwith a CV of 2.3% and the mean retention time for dehydronorke-tamine was 13.8 min with a CV of 2.0%. The mean relative retentiontime of ketamine and dehydronorketamine was 0.94 with a CV of1%. The mean capacity factor for ketamine was 1.07 with a CV of 4%and for dehydronorketamine was 0.95 with a CV of 2%.

In order to maintain sensitivity and limit zone dispersion, on-column focusing was applied to the sample by stacking it onto thehead of the column using 0.1% formic acid. During development ofthe approach, three different sized sample loops of 100 nL, 500 nLand 1 �L were investigated. The peak width and intensity for aninjection of an aqueous standard solution of ketamine (100 pg/mL)was recorded for each size of sample loop (data not shown). Thepeak width did not alter between the three loop sizes but the peakheight increased in proportion to increasing the sample loop size.When a small amount (5%) of acetonitrile was added to eitherthe standard solution solvent or to the stacking flow conditions,the ketamine peak would broaden to ∼20 s accompanied by peaktailing. This indicated that on-column focusing was being achievedusing aqueous conditions and provided that this was done, the use

Fig. 3. Selected ion chromatograms for the three ion transitions of ketamine froma 100 pg/mL standard solution.

M.C. Parkin et al. / J. Chromatogr. A 1277 (2013) 1– 6 5

Table 1Detection of ketamine and its primary metabolites in hair segments of volunteers. The time period (post administration) when the hair was collected is given in parenthesesalong with the mass of hair used for extraction from each segment.

Segment (proximal to distal)

A B C D E

Volunteer 1 (59 days) 24.9 mg 30.1 mg 26.2 mg 30.0 mg 28.14 mgKetamine + + + + –Norketamine + + + + –Dehydronorketamine + + – – –

Volunteer 2 (56 days) 23.8 mg 26.6 mg 41.3 mg 54.8 mg 47.4 mgKetamine + + + + +Norketamine + + + + +Dehydronorketamine + + + – –

Volunteer 3 (53 days) 19.2 mg 24.2 mg 27.6 mg 20.6 mg 18.3 mgKetamine + + + + +Norketamine + + + + +Dehydronorketamine + + + + –

Volunteer 4 (98 days) 28.9 mg 33.0 mg 34.9 mg 36.7 mg 42.1 mgKetamine – + + + +Norketamine + + + + +

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Dehydronorketamine – –

Compound detected; – compound undetected.

ig. 3 shows the three ion transitions of ketamine for a 100 pg/mLtandard solution. A linear response (r2 > 0.995) was observedcross the range of standards used to calculate the LOD (10 pg/mL,0 pg/mL, 100 pg/mL, 500 pg/mL and 1 ng/mL). The LOD based on a/N ratio >3 for all three ion transitions of each analyte were esti-ated to be 5 pg/mL for ketamine and norketamine and 10 pg/mL

or dehydronorketamine. For an injection volume of 1 �L this wasquivalent to a detection limit of 5 fg on-column for ketamine andorketamine and 10 fg on-column for dehydronorketamine. Thelank hair samples did not generate any interfering ions that coulde observed in the SRM transitions.

.3. Detection of ketamine and its primary metabolites in hairamples

Recently there have been a number of publications using LC–MSs the analytical probe for the detection of ketamine in hair samples25–27] whereas previously GC–MS was used [28–30]. Ketamine,orketamine and dehydronorketamine were detected in all volun-eer samples but not in all the sections of hair for each volunteers shown in Table 1. Any possibility of contamination due to inade-uate cleaning of the ball mill was addressed by pulverising knownlank hair samples in the mill between each volunteer and verifyinghat they were blank. Washing hair under aqueous conditions forecontamination purposes is thought to cause hair swelling whichan result in external contamination of drugs penetrating into theair matrix [31]. In order to avoid hair swelling, methanol was useds the wash solvent to remove any external contamination. Ultra-onication in the presence of methanol has been used to extractrugs from hair [32]. However, in this study, an initial investigation

nto the use of methanol and ultrasonication for extraction pur-oses was conducted (data not shown). Despite prolonged periodsf ultrasonication (>3 h), analytes were not detected in the extrac-ion solvent. Mechanical break up of the hair shafts using a ball mills described was successful in extracting ketamine and its metabo-ites from the hair and this was the process adopted for extraction.he methanol ultrasonication stage was adopted to externally washhe hair and the methanolic wash stage was confirmed blank. Fig. 4hows a typical selected ion chromatogram for one of the hair sec-

ions from the volunteers (segment C, volunteer 4). Each analyteas separated with very sharp peaks and retention times were

3.6 min, 13.8 min and 14.1 min for dehydronorketamine, norke-amine and ketamine, respectively. The long chromatographic run

+ + –

time and elution time of ketamine and its metabolites would makethe approach less suitable for screening purposes where there maybe a need for high sample throughput. However, its high sensitiv-ity makes the approach useful for confirmation work or where theanalytes are suspected to be found in low concentrations.

In a similar study, using conventional LC, the authors reporteda widening displacement of the ketamine-positive area in the hairshaft. They postulated that this may be due to sweat or sebum sec-retions that occur during the formation of the hair shaft [27]. Thehair shaft can also be continuously bathed in sweat as it grows outfrom the scalp, also contributing to drug incorporation. Likewise,in this work, the distribution of ketamine and its metabolites didnot appear to be localised to a specific region along the hair shaft.The drug and its metabolite (norketamine) were detected along a2.5 cm length in three out of the four volunteers and this broad-ening band was of a similar length to that reported by Xiang et al.[27].

3.4. Single particle frit and integrated electrospray emitter

To our knowledge, the detection of dehydronorketamine inhair samples is a novel and notable finding as the amount of thismetabolite present would be expected to be very low and could notarise from environmental contamination. Further work is requiredto quantitatively determine the amount of drug and metabolitespresent in these samples per mass of hair from the volunteers.However, this should not detract from the significance of the workpresented here as it represents a highly sensitive screening methodfor the detection of ketamine and its metabolites in hair. The pres-ence of a single 50 mg dose of ketamine was detected in hairsamples up to 98 days following administration.

The simplicity of the single particle frit provides a rapid method-ology for the preparation of 20 �m i.d. fused silica nanoflowcolumns and utilising the approach for the detection of drugs inhair proffers an advantage over the analysis of blood and urine inextending the window of detection. The columns are reusable andcan be used for >50 h of analysis time before blocking occurs. Thecolumns can also be made in batches of 10 or more at a time andcan be discarded when blocked and readily replaced.

The preparation of such narrow bore columns is not trivial andthe time taken to acquire the skills required for fritting and thenpacking the phase is unlikely to be available to analytical toxico-logy laboratories primarily dedicated to routine analysis of samples.

6 M.C. Parkin et al. / J. Chromatogr. A 1277 (2013) 1– 6

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evertheless, the approach of using a nanoflow column, with anntegrated electrospray emitter, is shown here to offer a largencrease in sensitivity that is particularly valuable in the forensicontext. To progress this application, ideally the preparation of sucholumns would be taken on by commercial suppliers of chromato-raphic products, particularly those that supply low flow pumps.his would help to establish the validity of this set-up in the foren-ic environment, where analysts have to work to strict reportingriteria. Commercial packing of nanoflow columns would hopefullyinimise between batch variability of chromatographic charac-

eristics, particularly retention times of target analytes. Anotherhallenge is to eliminate column blocking, which does occur, andurther investigation regarding sample preparation is necessary bute do not envisage this to be a major hurdle to be overcome. In the

nterim, this approach appears to be a valuable analytical probe foresearch investigations as to whether particular drugs and theiretabolites are present in alternative matrices in amounts thatould be missed by conventional approaches.

cknowledgements

This research was supported by a Wellcome Trust “Value in Peo-le” award (MCP) and a “Think Crime” grant (EP/C533437/1) fromhe Engineering and Physical Sciences Research Council (EPSRC),K. We thank Mrs Catherine Downie for the photographic imagesf the fritting process and Agilent for the loan of the Chip Cubeomponents.

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[

[[[

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