Analysis of meconium, nails and tears for determination of medicines and drugs of abuse

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Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

Analysis of meconium, nails and tears for determination of medicines and drugs of abuse Katarzyna Anna Madej This review covers methods for determination of medicines and drugs of abuse in three biological specimens – meconium, nails and tears – based on the literature since 1998. It starts with general descriptions of specimens, sample-collection methods and sample-preparation procedures. The key questions addressed relate to drug analysis in meconium, nails and tears. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Biological specimen; Drug; Drug analysis; Drug of abuse; Meconium; Medicine; Nail; Sample collection; Sample preparation; Tears

1. Introduction Katarzyna Anna Madej* Jagiellonian University, Faculty of Chemistry, Ingardena 3 Str., Krako´w, 30-060, Poland

*

Tel.: +48 12 663 22 57; Mob.: +48 502 600 372; E-mail: [email protected]

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In recent decades, growing interest has been noted in determination of drugs in alternative biological materials, mainly promoted by intensive development of highly sensitive and selective techniques, especially liquid chromatography with mass spectrometry (LC-MS) and LC with tandem MS (LC-MS2). The most important unconventional biosamples include hair, oral fluid, sweat, meconium, nails and tears. All these specimens present a crucial advantage (i.e. almost non-invasive collection). Furthermore, collection may be conducted under supervised conditions to prevent adulteration or substitution of samples, as this is essential in some analytical fields, (e.g., analysis for control of doping drugs or forensic analysis). Besides, in comparison with routinely-used urine or blood samples, some of these materials (e.g., hair, nails and meconium) are characterized by a larger detection window. This considerably increases their application range [e.g., workplace drug testing (hair) or prenatal exposure to drugs of abuse (meconium)]. Nowadays, oral fluid and sweat are also used in evaluating drug use

by drivers [1]. Moreover, unconventional biosamples constitute valuable evidence materials, which give information not available in routine forensic toxicological analysis. However, in spite of the sensitivity of existing methodologies, the chief problem in analyzing these materials remains the small amounts of sample available. A recently published book [2] and separate chapters in specialized books on analytical forensic toxicology have been dedicated to the state-of-the-art determination of drugs in some alternative specimens (e.g., hair, saliva/oral fluid and sweat [3,4] and meconium [3]). Comprehensive reviews on analysis of drugs in one selected alternative material (e.g., hair [5,6], oral fluid [7,8] and meconium [9]) have also been published. Chromatographic and/or capillary electrophoresis (CE) procedures for determination of one selected group of illicit drugs (e.g., methylenedioxyamphetamine derivatives [10] or cannabinoids [11]) in several alternative biological specimens have also been reviewed. Recently, there was a review on advances in chromatographic methods to detect drugs of abuse in alternative specimens (e.g., hair, sweat, nails, saliva and meconium) [12]. However, there is a lack of review papers on advances in analytical studies of meconium, nails and, especially tears, for determination of various classes of drugs, including licit and illicit drugs. This article focuses on determination of medicines and drugs of abuse in the three matrices mentioned above.

0165-9936/$ - see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.01.005

Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

2. Analyzing meconium [13,14], nails [15,16] and tears [17] 2.1. General characteristics 2.1.1. Meconium. Meconium is the first fecal matter passed by a neonate, and is commonly characterized by its dark-black color and lack of the odor of regular feces. It is formed between the 12th and the 16th weeks of gestation, and then accumulated and confined in the fetal bowels until birth. Meconium is a complex matrix comprising water, mucopolysaccharides, bile salts, bile acids, epithelial cells and other lipids, as well as residue of swallowed amniotic fluid. Its analysis allows detection of drugs or other substances to which the fetus was exposed in uterus during about the last 20 weeks of gestation. 2.1.2. Nails. The human nail plate is a keratin structure that comprises three layers:  the dorsal (upper layer);  the intermediate layer derived from nail matrix; and,  the ventral layer derived from nail bed. The nail plate overlays the nail bed (a non-cornified tissue), and, at the interface, nail-bed cells are carried distally by the nail plate during the growth towards the free margin. Nails grow in two different directions, length and thickness. The proliferation of the matrix involves a distal growth of nails at the rate of 0.1 mm/ day for fingernails and 0.03–0.04 mm/day for toenails. The thickening rate is constant and slow, with a mean value of 0.027 mm/mm length. Thickening is caused by formation of ventral layers by the nail bed during growth from the lunula (the proximal whitish and moon shape part of nails) to the free margin. Nail growth is also affected by different factors (e.g., age, climatic conditions, disease or malnutrition). Nail plate is almost completely formed by the 20th week of the fetal life. 2.1.3. Tears (lachrymal fluid). Lachrymal fluid impregnates the cornea, the conjunctiva and the nasolachrymal ducts. It ensures moisture of the cornea, allows blinking and enhances tear elimination. Lachrymal fluid is organized into a structured film comprising three different layers and it protects the conjunctiva and the cornea against physical and chemical agents. It contains many exogenous and endogenous compounds (e.g., H2O 98%, Na+ 134–170 mmol/L, K+ 20–40 mmol/L, HCO3 26 mmol/L, phosphorus 8–10 mmol/L and proteins 6– 10 g/L). Lachrymal fluid is constantly secreted by basal glands, but their secretion can be dramatically increased by physical and emotional stimulation. In humans, the lachrymal fluid is characterized by the following physiological parameters: volume 6–7 lL/min, basic flow about 1.2 lL/min and maximal capacity 30 lL without blinking.

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2.2. Sample collection 2.2.1. Meconium. Meconium sampling is easy and completely non-invasive. It is achieved by scraping the contents (minimum 0.5 g) of the soiled diaper into a special collection container. Studies indicate that drugs are stable in such container for up to two weeks at room temperature and for at least one year if frozen [3]. This material is characterized by a wide window for sample collection and can be sampled 1–5 days after birth. One issue that should be taken into account when collecting meconium is the possibility of urine contamination, which is likely to occur when a neonate evacuates drugcontained urine into a meconium-soiled diaper. 2.2.2. Nails. The sampling procedure of fingernails and toenails is rather simple. Nail samples are usually obtained by cutting the excess overhang of the nail plate using cosmetic nail clippers. The samples of each person examined are pooled and stored (e.g., in sealed plastic bags) at room temperature with limited light exposure until required for analysis. 2.2.3. Tears. Sampling tears is the chief problem to producing precise, reproducible analytical results. The two main procedures for collecting tears are: 1) direct sampling and 2) indirect sampling. Direct sampling comprises collecting tears with capillary tubes and requires previous stimulation, which facilitates withdrawal of lachrymal secretions and may be conducted in three main ways: (a) chemical (e.g., fumes of liquid agents, such as ethanol, formalin or ammonia); (b) physical stimuli (e.g., intensive light); or, (c) physiological stimuli (e.g., sneezing or yawning stimulation). An alternative procedure involves instilling various amounts of liquid (e.g., 20–100 lL saline solution), but this technique is rather limited to qualitative examinations. The main disadvantages of direct sampling are: (a) major dilution of tears induced by stimulation; (b) lack of a standardized time required to collect a sufficient volume of tears; and, (c) the difficulty of collecting samples from specific sites (e.g., under the eyelid). Indirect sampling uses absorbing supports that are very similar to Schrimer strips (classically used to diagnose dry-eye syndrome). These strips are made of cellulose filter paper and possess precise characteristics to promote good tolerance. The different components from this strip are generally released out by impregnating with an appropriate solvent (e.g., mobile phase when LC is the analytical technique). Total removal of the compounds may be facilitated by agitating with ultrasound. The liquid receiver can be frozen before analytical measurement.

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Indirect sampling is rapid (usually less than 5 min), easy and better tolerated than direct sampling (i.e. less risk of trauma). Standardization of the time necessary for tear collection promotes reproducibility of the analytical results. However, indirect sampling does not require preliminary stimulation; it is responsible for altering the epithelium and promoting leakage from plasma. 2.3. Sample preparation 2.3.1. Meconium. Taking into account matrix complexity, meconium analysis generally requires a thorough, preliminary clean-up procedure, including solid-phase extraction (SPE) and sometimes liquid-liquid extraction (LLE), prior to any analytical assays. In order to facilitate meconium-sample handling, a weighed amount of meconium is homogenized in methanol, a mixture of methanol and acetonitrile or a suitable buffer, and centrifuged. (Instead of the term ‘‘homogenization’’ some authors use the term ‘‘emulsification’’ or even ‘‘extraction’’). The supernatant is then removed and evaporated, and the residue is reconstituted with a suitable buffer and then subjected to an appropriate extraction procedure. In some cases, alkaline or enzymatic hydrolysis is necessary to release target compounds from more complex organic combinations (e.g.,

Meconium sample

glucuronides). When using gas chromatography (GC) as the determination method, a derivatization step may also be needed. Fig. 1A shows the key steps in preparing a meconium sample for analysis. 2.3.2. Nails. Fig. 1B shows the four key steps in preparing nail samples: 1) decontamination; 2) cutting into small segments; 3) alkaline, acidic or methanolic digestion (hydrolysis); and, 4) extraction (usually LLE). In some cases, SPE (together with LLE) as well as derivatization (when GC is used) may be applied. For surface decontamination, nails are usually washed in an appropriate mixture of reagents (e.g., water, methanol, acetone or surfactant SDS) using an ultrasonic bath. 2.3.3. Tears. Preparation of tears for analysis is not complicated, and often only one step is required (Fig. 1C). A tear sample is usually prepared in one of the three ways: 1) dilution with an appropriate solvent; 2) precipitation of proteins (e.g., with acetonitrile or perchloric acid); or, 3) LLE.

Nail sample Tear fluid sample Decontamination

Homogenization Cutting into small pieces

Precipitation of proteins

Dilution

Hydrolysis Digestion

LLE

LLE LLE SPE

SPE

Derivatization

Derivatization

Analytical measurement

Analytical measurement

A

B

Analytical measurement

C

Figure 1. General sample preparation procedures for: meconium (A), nails (B) and tears (C). The dashed lines indicate steps that are not the basic ones and may be used in only some cases of analyses.

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Drugs

Sample amount (g)

Sample preparation (the main steps)

Extraction recovery (%)

Analytical method

Limit of detection (LOD) or limit of quantification (LOQ)

Precision (%, RSD)

Ref.

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1.0

1) homogenization in 100% methanol 2) SPE 3) derivatization with BSTFA (N,O-bis-trimethylsilyltrifluoroacetamide)

mean recovery was 58 ± 27 and in the range 26.5–98 recovery rates for cocaine, benzoylecgonine, codeine, morphine and methadone were 82, 91, 72, 62 and 59, respectively.

1) HPLC-UV 2) GC-MS

the minimum detectable drug concentration was 200 ng/mL

-a

[18]

0.5

1) homogenization in 25% methanol/acetonitrile 2) SPE

CEb-UV

LOD 5 lg/g LOQ 10 lg/g (for all examined barbiturates)

mean precision (between-run) was 3.36

[19]

Stimulants: nicotine, cotinine and caffeine

2.0

HPLC-DAD

LOD 10 ng/mL (for all tested alkaloids)

-a

[20]

Stimulants: cotinine (a metabolite of nicotine)

0.5–1.0

1) homogenization in phosphate buffer (pH 8.0) 2) LLE with chloroform 3) SPE 1) homogenization in methanol 2) hydrolysis with KOH 3) SPE

in optimal conditions extraction efficiency ranged from c.a. 100 (for pentobarbital, secobarbital and amobarbital) to 22.9 (for phenobarbital) nicotine 90 (only for spiked samples) cotinine 85 caffeine 92 -a

Enzyme immunoassay (EIA)

-a

-a

[21]

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Local anesthetics: lidokaine, mepivacaine, procaine, bupivacaine, ketamine Analgesics: mepiridine, codeine, dextromethorphan Antihistamines: diphenhydramine, terfenadine, hydroxyzine, promethazine Adrenergics: tyramine Antibacterial: methenamine Carditonic: heptaminol Anticonvulsants: beclamide Antidepressants: sertraline Expectorants: guaifenesin Illicit drugs: cocaine morphine methadone amphetamine Stimulants: nicotine (cotinine – a metabolite of nicotine) Barbiturates: pentobarbital, mephobarbital, phenobarbital, secobarbital and amobarbital

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Table 1. Determination of drugs in meconium

Drugs http://www.elsevier.com/locate/trac

Sample amount (g)

Sample preparation (the main steps)

Extraction recovery (%)

Analytical method

Limit of detection (LOD) or limit of quantification (LOQ)

Precision (%, RSD)

Ref.

Illicit drugs: opiates (6monoacetymorphine, morphine, morphine-3glucuronide, morphine-6glucuronide, codeine) cocaine (metabolites: benzylecgonine, cocaethylene) Illicit drugs: cocaine and fifteen cocaine metabolites

1.0

1) homogenization in methanol or 0.01M ammonium hydrogen carbonate buffer 2) SPE

mean recovery was 84.2 ± 4.35 and in the range 74–90

LC-ESI-MS

Mean LOD was 0.94 ng/g

mean precision was 7.6

[22]

0.5

1) homogenization in methanol 2) SPE

at the low concentration (12 ng/g) the recovery for the metabolites was in the range 42.29–59.11

LC-MS-MS

LOQ was between 1 and 5 ng/ g

[23]

Methadone and its metabolites (EDDP: 2ethylidene-1,5 dimethyl-3diphenylpyrrolidine, EMDP: 2-ethyl-5-methyl-3,3diphenylpyraline and methadol) Buprenorphine, norbuprenorphine and their glucuronide conjugates

0.5

1) homogenization in methanol 2) SPE

-a

LC-APCI-MS2

LODs were: 1.0 ng/g for methadone, EDDP and EMDP, and 2.5 ng/g for methadol

at the low concentration level (5 ng/g) precision (interday) was ranged from 0.60 to 19.88 mean precision (inter-day) was 14.23

0.25

1) homogenization in sodium acetate buffer (pH 5.1 2) enzymatic hydrolysis (Glusulase) 3) SPE 1) homogenization in methanol 2) enzymatic hydrolysis (bglucuronidase) 3) SPE 4) derivatization with Nmethyl-N-trimethylsilyltrifluoroacetamide (MSTFA) 1) homogenization in 17 mM methanolic HCl 2) SPE

mean recoveries were: 87.3 for buprenorphine and 84.5 for norbuprenorphine

LC-APCI-MS2

LOD was 20 ng/g for all analytes

mean recoveries were: 99.8 for 9-THC and 98.4 for 11OH-THC

Twodimensional (2D) GC-MS

LOD was 5 ng/g LLOQc was 10 ng/g

mean recoveries in the range 61.1–87.2 for different analytes

LC-APCI-MS

LOD was 1.0 ng/g LOQ ranged from 4.0 to 5.0 ng/g

mean precision (inter-day) ranged from 1.6 to 15.0

[27]

mean recovery in the range 63.3–80.6

LC-APCI-MS2

LLOQc in the range 1.25–40 ng/g

precision was less than 14.2

[28]

1.0

Illicit drugs: amphetamine(AP), methamphetamine(MA) methylenedioxy derivatives Illicit drugs: 10 amphetamine-, methamphetamine- and 3,4methylenedioxymetha mphetamine-related analytes

1.0

a b c

No data were given. Capillary electrophoresis. Low limit of quantification.

1.0

1) homogenization in 17 mM methanolic HCl 2) SPE

[24]

[25]

[26]

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Illicit drugs: Cannabinoids: 9-carboxy-11nor-D9- THC (9-THC) and 11hydroxy-D9-THC (11-OHTHC)

mean precision (inter-day) was 9.35 (for buprenorphine) and 8.17 (for norbuprenorphine) mean precision 8.43 for 9-THC and 8.16 for 11-OH-THC

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Table 1. (continued)

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Sometimes a derivatization step may be needed when GC is used for the determination.

3. Determination of drugs in meconium, nails and tears 3.1. Meconium (Table 1) Meconium analysis is a very sensitive tool for assessing the risk of gestational exposure to drugs and other xenobiotic agents in newborn infants. A study provided evidence of the exposure of the fetus in American women to a wide spectrum of illicit drugs and commonly prescribed medicines [18]. This study covered 98 randomlyselected infants and demonstrated that 82.7% infants tested positive for xenobiotics. In the infants who tested positive, 1–9 drugs were identified, including local anesthetics (30%), illicit drugs (11%) [e.g., cocaine, morphine, methadone and amphetamine (AM)], analgesics (10%), and other drugs (<10%) (e.g., antihistamines, antidepressants, adrenergics, cough medications, anticonvulsants, antibacterial agents and cardiotonics). CE with ultraviolet detection (CE-UV) was used for simultaneous determination of five barbiturates (i.e. pentobarbital, mephobarbital, phenobarbital, secobarbital and amobarbital [19]). Some commonly prescribed drugs [e.g., aspirin or acetaminophen (paracetamol)], stimulant caffeine, phenytoin (anticonvulsant) and atropine did not interfere with the determination of the barbiturates. Meconium was also analyzed to assess fetus exposure to tobacco smoke [20,21]. An HPLC method with diodearray detection determined nicotine and its metabolite, cotinine, and caffeine, in meconium [20]. The three compounds were eluted in <9 min, but, under these conditions, cotinine and caffeine were still not completely separated. When 30 real meconium samples were analyzed, 11 samples were positive for cotinine (range 20–86 ng/g) and 27 were positive for caffeine (range 10–45 ng/g). No nicotine was found in the samples because of its rapid metabolism into cotinine. Cotinine was found in meconium obtained from newborns whose mothers were active or passive (i.e. exposed to secondhand smoke) smokers, but no cotinine was detected in meconium obtained from infants whose mothers did not smoke. The authors recommended to subject extracts of meconium samples to analysis as quickly as possible, and stated that concentrations of alkaloids in meconium had remained without change for at least one week when the samples were stored at 18C. Dempsey et al. [21] compared the results of cotinine detection in 102 meconium samples using two methods of sample preparation: 1) a routine non-hydrolysis extraction procedure for screening of drugs of abuse; and,

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2) the same extraction procedure with previous hydrolysis. The extracts from these two methods were analyzed by a micro-plate enzyme immunoassay (EIA) method that was highly specific for cotinine. From the non-hydrolyzed samples, 33% were positive for cotinine, while 79% of the hydrolyzed samples were cotinine-positive. The authors stated that the results obtained supported the theory that ‘‘cotinine forms Schiff base bonds with amino acids within meconium and hydrolysis of the specimen cleaves such bonds’’. In most cases of meconium analysis, there were procedures for screening and quantification of drugs of abuse with their metabolites (e.g., opiates [22], cocaine [22,23], methadone [24], buprenorphine [25], cannabinoids [26] and AMs [27,28]). Pichini et al. [22] described a procedure based on LC-MS for determination of opiates (morphine, 6-monoacetylmorphine, morphine-3glucuronide, morphine-6-glucuronide and codeine) and cocaine with metabolites (benzoylecgonine and cocaethylene) in meconium. The authors stressed that the metabolites that they detected (morphine glucuronides or cocaine metabolites) in the meconium samples examined were not (or not in all cases) found in this material by other investigators. An LC-MS2 method quantified cocaine and its 15 metabolites in meconium [23]. Under optimal conditions, chromatographic separation of the compounds of interest was achieved in <17 min. The ability of the method to study cocaine exposure in neonates was checked by analyzing suspected cocaine-positive meconium samples, and, of the 22 samples, only one did not show the presence of cocaine or any metabolite of cocaine. On the basis of the results, the authors concluded that ecgonine (one of the cocaine metabolites tested) seemed to be the most promising compound as a diagnostic marker for neonatal cocaine exposure, but a group of eight metabolites was identified in at least 20 of 21 positive samples that appeared to be of highest utility for determining cocaine exposure. Two LC-MS2 methods were developed for determination of methadone [24] and buprenorphine [25], which are used for treatment of opiate addiction during pregnancy, together with their metabolites. Choo et al. [24] reported an LC-APCI-MS2 method for simultaneous quantification of methadone with two metabolites [i.e. 2ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP)] and semi-quantification of methadol (third metabolite) in a meconium matrix. Separation of the four compounds of interest was performed in <11 min. The usefulness of the method developed was demonstrated by analyzing a meconium specimen from an infant whose mother was maintained on methadone for 19 weeks of gestation. The meconium examined contained 2492 ng/g of

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methadone, 13.188 ng/g of EDDP and 27.00 ng/g of EMDP. An LC-APCI-MS2 method quantitatively analyzed buprenorphine, norbuprenorphine and glucuronidated conjugates in meconium [25]. The method was applied for analysis of a meconium specimen from an infant born to a woman treated with up to 20 mg/day buprenorphine for the last 12 weeks of pregnancy. Total and free buprenorphine concentrations were 123.8 ng/g and 101.6 ng/g, respectively. Norbuprenorphine concentrations were higher, 719.3 ng/g for total drug and 712.6 ng/g for the unconjugated compound. According to the authors of the two methods mentioned above, the proposed procedures will help to elucidate the relationship between drug concentrations and infant outcome. Marin et al. [26] developed a procedure for confirming cannabinoids in meconium. Two-dimensional GC with MS detection [(2D)GC-MS] was employed for determination of 9-carboxy-11-nor-D9-THC (9-THCA) and 11hydroxy-D9-THC (11-OH-THC). This technique reduced interference and carry over from the meconium matrix and turned out to be superior to the existing conventional GC-MS method (single column). The proposed method was applied to 10 patient specimens that had previously failed to confirm cannabinoids due to interferences using conventional GC-MS method, and 9THCA was detected and quantified in all 10 samples. Two LC-MS2 methods determined AM, methamphetamine (MA) and methylenedioxy derivatives in meconium samples [27,28]. AM, MA, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 4-hydroxy-3-methoxymethamphetamine and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine were simultaneously detected and quantified in meconium specimens [27]. The feasibility of the proposed method was verified by using it to analyze meconium samples belonging to mothers who declared heavy MA abuse during pregnancy. Analysis of these samples revealed high concentrations of both MA and AM, without presence of any methylenedioxy derivative. Experimental studies also proved a long period (over one year) of AM and MA stability in meconium during storing the samples at 20C. An LC-APCI-MS2 method was fully validated for simultaneous quantification of 10 AM-, MA- and MDArelated compounds in meconium [28]. All analytes were eluted within 15 min. The method developed was applied to a meconium specimen obtained from a neonate following exposure in uterus to AM-related compounds. The authors stated that three new biomarkers of AM-related compounds exposure – p-hydroxymethamphetamine, norephedrine and 4-hydroxy-3-methoxymethamphetamine – were identified and quantified for the first time in a meconium matrix.

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3.2. Nails (Table 2) A variety of licit drugs (b-blockers, sedatives, anticoagulant agents, antidepressants and antipsychotics) and illicit drugs (cocaine, cannabinoids, morphine and AMrelated compounds, including their metabolites) were detected and determined in nails. The possibility of determination of b-blocker drug atenolol in nails was studied employing LC-MS by means of electrospray ionization (LC-ESI-MS) [29]. The method was applied to determination of the atenolol level in nails of patients who had taken this medicine for 6–12 months. The presence of atenolol was ascertained in all samples examined (30) and its mean concentration level in nails (0.155 ng/mg) was lower compared to the level determined in the hair samples (1.73 ng/mg), which were simultaneously taken from the same patients. Irving et al. [30] described a screening method for nine sedatives [zopiclone and eight benzodiazepines (alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam and triazolam)] and their selected metabolites in human nails and hair employing LC-MS2. The study established that drugs tested were incorporated into nails at levels similar to those in hair. The levels of the drugs in subjects on regular medication suggested that a single dosage of some of these drugs could be detectable. The authors emphasized that the presence of both parent drug and metabolite(s) in nails can provide strong supporting evidence in an increasing number of allegations of drug-facilitated sexual assaults. The stability of ticlopidine (anticoagulant agent) in human blood, hair and nails was studied using LC-ESIMS [31]. The influence of light, temperature and time of storage on the concentration of ticlopidine was examined. The samples were analyzed before storage, after 14 days and again after 60 days. The results obtained demonstrated that ticlopidine was a very stable substance and suggested that hair and nails can be stored at room temperature, but preferably should be protected against light. A few reports described determinations of various psychotropic drugs [32,33]. The possibility of using fingernails and toenails for determinations of psychotropic drugs (e.g., haloperidol [32] or flupentixol [33]) was studied by LC-ESI-MS. Antipsychotic haloperidol was determined in fingernail and toenail samples originating from individuals who had been administered this medicine at least 6 months prior to sample collection. The materials analyzed demonstrated the presence of haloperidol in the following amounts:  fingernails – 67.3 ± 6.49 pg/mg; and,  toenails – 98.9 ± 9.14 pg/mg. Determinations of antidepressant flupentixol were performed in fingernail and toenail samples obtained from patients who had been administered this drug for at least 12 months before sample collection. The nails were

Drug

Sample amount (mg)

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Atenolol (b-blocker)

100

Benzodiazepines: alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazolam and zopiclone

c.a. 50

Ticlopidine (anticoagulant agent)

100

Haloperidol (antipsychotic drug)

100

Flupentixol (antidepressant and antipsychotic drug)

P 50

Phenothiazines: promazine, chlorpromazine, levomepromazine, thioridazine Tricyclic and tetracyclic antidepressants: amitriptyline, mianserine, clomipramine Cocaine and its metabolites (illicit drugs)

-a

Cannabinoids (illicit drugs)

7–50

2.5–21.5

Sample preparation (the main steps)

1) decontamination by water and then acetone 2) cutting into 1–2 mm segments 3) digestion with 0.1 M HCl 4) LLE with dichloromethane 1) decontamination by ethanol 2) cutting into 1–2 mm pieces 3) digestion with the mixture of trifluoroacetic acid and methanol (1:50) 4) LLE with dichloromethane

1) decontamination by water and methanol 2) cutting 3) LLE with n-hexane 1) washing with water and then acetone 2) cutting into 1 mm segments 3) digestion with 1 M NaOH 4) LLE with n-hexane/chloroform (7:3, v/v) 1) decontamination with water and then with acetone in ultrasonic bath 2) cutting 3) digestion with 1 M NaOH 4) LLE with n-hexane 1) digestion with 1 M NaOH 2) LLE with chloroform from pH 8–10 solution

Analytical method

Limit of detection (LOD) or limit of quantification (LOQ)

Precision (%, RSD)

Ref.

-a

LC-ESI-MS

LOD 0.025 ng/mg

precision (inter-day) was 6.9

[29]

-a

LC-MS2

LOD 0.01–0.60 pg/mg

[30]

>80

LC-ESI-MS

-a

precision measured for four compounds (diazepam, nordiazepam, zolpicone and N-desmethyl zopiclone) in the range 5–59 -a

-a

LC-ESI-MS

LOD 1.0 ng/mg LOQ 2.5 pg/ml

precision in the range 3.1–16.5

[32]

-a

LC-ESI-MS

LOD 1.0 pg/mg LOQ 2.0 pg/ml

precision in the range 4.2–9.1

[33]

-a

LC-MS

-a

-a

[34]

-a

GC-MS

-a

[35]

> 81

RIA and GCMS

LOD 0.10 ng/mg and 0.25 ng/mg for cocaine analytes, and ecgonine ethyl ester and norbenzoylecgonine, respectively LOD < 0.1 ng/mg

-a

[36]

[31]

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1) cutting 2) washing with methanol 3) extraction with methanol (heating under reflux at 40C for 16 h) 4) SPE 1) decontamination by sonication with SDS, water and methanol 2) alkaline hydrolysis with 1 M NaOH 3) LLE with ethyl acetate 4) derivatization with BSTFA and 1% TMCS

Extraction recovery (%)

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Table 2. Determination of drugs in nails

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Table 2. (continued) Drug

Sample amount (mg) 3.0–96.0

Illicit drugs: morphine, 6-acetylmorphine and cocaine

50.0

Illicit drugs: Amphetamine-type stimulants: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphe tamine Cannabinoids: 9-carboxy-11-nor-D9THC (9-THC) and 11-hydroxy-D9THC (THCCOOH)

30.0

a b

No data were given. Electrochemical detection.

Extraction recovery (%)

Analytical method

Limit of detection (LOD) or limit of quantification (LOQ)

Precision (%, RSD)

Ref.

1) decontamination by sonication with SDS, water and methanol 2) alkaline hydrolysis with 1 M NaOH 3) LLE with dichloromethane:dichloroethane:heptane (19:18:63) 1) decontamination with methylene chloride 2) acid hydrolysis with 37% aq. HCl 3) LLE with chloroform/2-propyl alcohol solution (3:1, v/v) 4) SPE 5) derivatization with propionic anhydride 1) decontamination with water and methanol 2) hydrolysis with 1.0 M NaOH 3) LLE with ethyl acetate and re-extraction with n-hexane/ethyl acetate 4) derivatization with MSTFA

80.5 (RIA) 86.3 (HPLC)

RIA and HPLC-EDb

LOD 0.05 ng/mg

-a

[37]

-a

GC-MS

LOQ 0.1 ng/mg for morphine, 6-acetylmorphine and cocaine

-a

[38]

74.0–94.8

GC-MS

LOD < 0.056 ng/mg LOQ < 0.2 ng/mg

precision (inter-day) was 6.3

[39]

Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

Morphine (an illicit drug)

Sample preparation (the main steps)

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taken 4, 6, 7, 8 and 10 months after administration of this medicine was discontinued, and, 10 months after discontinuation of therapy, flupentixol was no longer found in nails. Various psychotropic drugs [e.g., tricyclic/tetracyclic antidepressants (amitryptyline, clomipramine and mianserine) and antipsychotic phenothiazines (chlorpromazine, levomepromazine, thoridazine and promazine)] were also determined in biological materials (blood, hair and nails) originating from victims of suicide by hanging [34]. In nail samples, the following amounts were confirmed:  amitriptyline (0.5–65.1 ng/mg);  mianserine (2.8–4.4 ng/mg);  chlorpromazine (3.3–20.6 ng/mg);  levomepromazine (3.3 ng/mg); and,  promazine (26.0 ng/mg). The usefulness of nails as an alternative matrix to blood and urine for detecting illicit drugs exposure was also demonstrated in procedures for determinations of cocaine, cannabinoids, opiates and MA-related compounds, including their metabolites. Fingernail and toenail samples obtained from 18 suspected cocaine users were subjected to qualitative and quantitative analysis for nine cocaine analytes (anhydroecgonine methyl ester, benzoylecgonine, cocaine, cocaethylene, ecgonine ethyl ester, ecgonine methyl ester, m-hydroxybenzoylecgonine, norbenzoylecgonine and norcocaine) by GCMS [35]. Cocaine analytes were present in 14 (82.3%) of subjects, while only 5 (27.7%) had been found positive in conventional post-mortem analysis. Cocaine and benzoylecgonine were the predominant compounds found in all positive nail specimens, with cocaine present at a concentration 2–10 times greater than benzoylecgonine. Concentration of cocaine analytes in fingernails was generally greater than in toenails, but external contamination should be taken into account. Lemos et al. [36] examined fingernail clippings as alternative specimens for detection and quantification of cannabinoids. The nail samples, after appropriate treatments, were analyzed by two methods [i.e. radioimmunoassay (RIA) and GC-MS]. Cannabinoids were found to be present in all six cases that were analyzed for this group of illicit drugs by RIA with mean concentration of 1.03 ng/mg. Using GC-MS, D9-tetrahydrocannabinol (9THC) was determined in 11 of the 14 examined nail hydrolysates extracted under basic pH with mean 9THC concentration of 1.44 ng/mg. The major metabolite, 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid (THCOOH), was detected in 2 of 3 fingernail hydrolysates extracted under acidic pH with average concentration of 19.85 ng/mg. The positive RIA and GC-MS results were obtained 6–9 months after nail collection. Lemos et al. [37] also evaluated the usefulness of fingernails as analytical specimens in identifying and quantifying morphine in heroin users. An RIA method

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was used for screening and an HPLC method for confirming morphine. Positive RIA results were obtained with nails from 25 of the 26 heroin users with mean morphine concentration of 1.67 ng/mg. HPLC results were positive for 22 of the 26 nail samples with mean morphine concentration of 2.11 ng/mg. Based on the results obtained, the authors concluded that nails could become a powerful alternative to hair for detecting past heroin use in forensic cases. In a comparable study, 18 post-mortem toenails and hair samples obtained from drug abusers were analyzed for presence of opiates and cocaine [38]. The results revealed that both cocaine and morphine were more concentrated in toenails than in hair. Mean concentrations were 0.99 ng/mg (toenails) versus 0.48 ng/mg (hair) for cocaine and 1.27 ng/mg (toenails) versus 0.79 ng/mg (hair) for morphine. Instead, 6-acetylmorphine (6-MAM) showed no significant variations between its concentrations in toenails (0.46 ng/mg) and in hair (0.50 ng/mg). The authors suggested that the results indicated a different mechanism of incorporating drugs in nails and hair, but there were sufficient positive matches between hair and toenail analysis. Toenails may therefore be used to complement or to provide an alternative to hair samples in forensic analysis. A GC-MS method for simultaneous detection and quantification of four AM-type compounds (AP, MA, MDA and MDMA) and two cannabinoids (9THC and THCCOOH) in fingernails was also developed [39]. The method was applied for analysis of nine fingernail samples obtained from abusers of illicit drugs. MA was the most frequently detected drug in association with its major metabolite – AM – and 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid was the second most frequently detected. The concentration ranges of MA, AP and THCCOOH were 0.10–1.41 ng/mg, 0.12–2.64 ng/ mg and 0.20 ng/mg, respectively. In contrast to the previous study [36], the presence of the parent drug (9THC) of THCOOH was not confirmed. 3.3. Tears (Table 3) Two antihistaminic drugs (i.e. dimethindene and cetirizine) were determined in human tears. A simple, fast HPLC-UV method quantified dimethindene in tears [40]. The tear samples (4 lL) were diluted with 0.01 M hydrochloric acid-n-propanol mixture to prevent irreversible adsorption of dimethindene, and then directly injected onto a CN column. The pharmacokinetics of cetirizine in tears after a single oral dose was also investigated [41]. Concentrations of cetirizine in both tears and serum taken from 40 volunteers with allergic conjunctivitis were determined at various times after oral administration of 10 mg dose to obtain the concentration-time curves up to 2 h. The analyses were carried out using HPLC-UV. For confirmation of cetirizine identity, ion-trap MS analysis was http://www.elsevier.com/locate/trac

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Drug http://www.elsevier.com/locate/trac

Sample amount [ll]

Sample collection

Dimethindene (antihistamine drug)

4

-a

Cetirizine (antihistamine drug)

50

using a capillary tube

Acetaminophen (paracetamol)

50

Valproic acid (antiepileptic drug)

c.a. 4

using a glass microcapillary hematocrit tube using Schirmer tear test strips

Docetaxel (antineoplastic agent)

50 (volume of 15–30 ll was analyzed) 50

using polyester rods

Sample preparation (the main steps) diluted with 0.01 M HCl - n-propanol, 95:5 (v/v) precipitation of proteins with 25% perchloric acid -a

Extraction recovery (%)

Analytical method

-a

HPLC-UV

-a

HPLC-UV and MS (for identification of the drug) Enzyme immunoassay (EIA)

-a

Limit of detection (LOD) or limit of quantification (LOQ)

Precision (%, RSD)

Ref.

LOD 2.4 ng/mL LOQ 12.0 ng/mL (312 pg/lL in the undiluted tear sample) -a

1.2–4.0

[40]

27–45

[41]

-a

-a

[42]

Ranged from 93.1 to 102.5

GC-ECb-NCIc-MS

10 pg per injection (1ll)

-a

[43]

-a

LC-MS2

10 ng

-a

[44]

-a

LLE with methyl tertbutyl ether

varied from 95.6 to 108.8

HPLC-EDd

0.1 mg/L for AZI and 0.2 mg/L for both ADES and NDES

<9.90

[45]

1

using a glass capillary

-a

LC-MS2

1 lg/mL

0.8–5.8

[46]

Besifloxacin (fluoroquinolone antimicrobial agent)

10–20 mg

using a Schrimer tear test strips

mean recovery was 93.7

LC-MS2

2.00 ng/mL

precision in the range 0.9–5.8

[47]

Thiamphenicol (antibiotic)

5

using a small plastic vial

mean recovery was 94.8

LC-MS

LLOQe 0.032 ng/mL

<8.3

[48]

Pradofloxacin (tetracycline antibiotic) and doxycycline (8cyanofluoroquinolone antimicrobial agent)

10

using a filter paper strip with one roundshaped ending

diluted with the mobile phase (5 mM pentafluoropropionic acid/ acetonitrile) LLE with 5 mM ammonium formate buffer (pH 3.25) precipitation of proteins with acetonitrile LLE using mixture of acetonitrile/water (1:1, v/v) with 2ml/L trifluoroacetic acid and 0.77g/L ammonium acetate

-a

LC-MS2

0.1 lg/mL

mean precision 20.85

[49]

Azithromycin (antimicrobial agent, AZI) and two of its metabolites: 9a-Ndesmethylazithromycin (ADES) and Ndesmethylazitromycin (NDES) Tobramycin (aminoglycoside antibiotic)

a

No data were given. Electron capture detection. c Negative chemical ionization. d Electrochemical detection. e Low limit of quantification. b

Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

derivatization to pentafluorobenzyl ester with DMF, PFBB and N,N-diisopropylethylamine -a

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Table 3. Determination of drugs in tears

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performed. Mean cetirizine concentrations in serum were in the range 79–105 lg/L, while its mean concentrations in tears were in the range 70–96 lg/L. The pharmacokinetics study indicated that a single daily administration of cetirizine was generally suitable for treatment of allergic conjunctivitis while only more severe cases required a twice daily administration. There was a comparable study of acetaminophen (paracetamol) concentration levels in human tears and serum, 1 h and 2 h after ingesting 1.5 g paracetamol [42]. Paracetamol levels were measured in tears and serum samples, simultaneously taken from 10 healthy volunteers. The samples were analyzed by homogenous enzyme immunoassay (UV spectrometry using Diagnostic ReagentsÕs Acetaminophen Tox EIA Assay). There was a strong correlation between paracetamol levels in serum and in tears 1 h and 2 h after ingestion, and the ratios of tear/serum paracetamol levels were 0.77 ± 0.21 and 0.81 ± 0.25, respectively. The authors emphasized that these results were valid for healthy individuals after administration of a therapeutic paracetamol dose, and further pharmacokinetic studies should be requested for poisoned patients. Nakajima et al. [43] presented a sensitive GC-electron capture negative chemical ionization-MS (GC-ECNCI-MS) method for assessing valproic-acid concentrations in tears in therapeutic drug monitoring of patients with epilepsy. However, an additional derivatization step converting valproic acid into its pentafluorobenzyl-ester derivative and further extraction with n-hexane were needed. Esmaeli et al. [44] performed a study to test the hypothesis that docetaxel (an antineoplastic agent widely used for treatment of metastatic or locally advanced breast cancer) might be secreted in tears. Secretion of docetaxel in tears would suggest an adverse effect of the drug. Two samples of tears were collected from six patients – one before infusion to serve as a control sample – and another sample within 30 min after the end of the 1-h docetaxel infusion. Tear volumes in the range 15–30 lL (obtained with help of two polyester rods) were directly injected into a binary HPLCMS2 system. A study with the plasma of all 6 patients showed that the docetaxel concentration in this biological fluid was several times greater than that in tears from these patients. These findings suggested that only a small part of the administered dose of docetaxel (the unbound portion of the drug to plasma proteins) had access to the tears. Several LC methods determined antibiotics and antibacterial agents in human [45–47], rabbit [48] and cat [49] tears. Azithromycin (AZI, a 15-membered ring azalide structurally related to erythromycin) and its two metabolites [9a-N-desmethylazithromycin (ADES) and N-desmethylazithromycin (NDES)] were measured in

Trends

human tears and plasma collected from trachoma patients receiving AZI [45]. The compounds of interest were determined by HPLC using electrochemical detection. The study showed that the concentration of AZI in tear samples was more than 10 times greater than in plasma at all times measured, with the ratio of the concentration of AZI in tears increasing as a function of time. A rapid LC-MS2 method determined tobramycin in 1 ll of human tears [46]. The samples were taken into precision glass capillaries and prepared in physiological saline (quality-control samples). After checking the volume of the sample, the capillary was put into a Pasteur pipette for sample transfer to vial containing the appropriate amount of the internal standard in 150 lL of the mobile phase. The authors stated that the proposed method had been used successfully to analyze 100 samples of human tears from a clinical study. Arnold et al. [47] developed an LC-MS2 method for the quantification of besifloaxacin in human tears using artificial tears as a surrogate matrix. The method was successfully used in a study of tear samples obtained from 64 healthy subjects following ocular administration of 0.6% besifloaxacin HCl in both eyes. Maximal besifloaxacin tear levels (610 ± 540 lg/g) and the lowest concentrations of this drug (1.60 ± 2.28 lg/g) were observed in tear samples collected 24 h after dosing. The stability study of besifloaxacin showed that it was stable for at least 197 days when the samples were stored at 20C. A highly sensitive LC-MS method in negative selectedion monitoring (SIM) mode, with a lower limit of quantification of 0.032 ng/ml, determined thiamphenicol (a broad-spectrum bacteriostatic antibiotic) in rabbit tears [48]. A simple sample procedure involved only organic solvent (acetonitrile) precipitation. The method was successfully applied in pharmacokinetic studies in rabbit of thiamphenicol in situ forming gel. A comparative study of pharmacokinetic properties of pradofloxacin (a novel 8-cyanofluoroquinolone developed for treatment of bacterial infections in cats and dogs) and doxycycline (tetracycline antibiotic) used turbulent flow chromatography-MS2 (TFC-MS2) [49]. Following administration, the concentration-time profiles of these two drugs in serum, saliva and tears of cats were determined. This study revealed high concentrations of pradofloxacin in saliva (Cmax = 6.3 ± 7.0 lg/mL) and tears (Cmax = 13.4 ± 20.9 lg/mL), when compared concentrations of doxycycline in these fluids. In tears, doxycycline was found in concentrations close to the limit of quantification in this specimen and doxycycline was not detectable in saliva. The authors concluded that pradofloxacin was more promising than doxycycline with respect to treatment of respiratory tract and conjunctival infections.

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4. Conclusions and outlook Meconium, nails and tears belong to alternative biological materials, which are not widely used for determination of drugs. One of the main reasons for this is the requirement of expensive, highly-sensitive and highlyselective methodologies. Most cases of analyzing these specimens employed LC-MS and LC-MS2 techniques. Sometimes, GC-MS, usually with a derivatization step, was used, while other techniques (e.g., HPLC-UV, HPLCED or CE-UV) were exceptional. Other analytical problems involving analysis of unconventional materials indicate that it is best to choose an appropriate sample-preparation procedure. Meconium has the most complex matrix of the three types of specimen. This matrix contains proteins, lipids and pigments at high concentration levels, so it should be prepared for analysis using a multi-step cleaning procedure, including homogenization and SPE, which is sometimes preceded by LLE. The nail matrix, as the hair matrix, mainly comprises keratin. It also requires a lengthy preparation procedure comprising decontamination, cutting into small pieces, hydrolysis (digestion) and, usually, LLE. Tears have a relatively uncomplicated matrix, which, in some cases of drug analysis, may be easily prepared – just by diluting with an appropriate solution or protein precipitation with an organic solvent. However, in many cases, LLE has been used and, in the one reported case of tear analysis, the derivatization step was followed by LLE. Studies on the stability of the drugs in the three biological matrices were also performed and revealed that these compounds were stable in nail matrix stored at room temperature with limited light exposure. The drugs examined were also stable in meconium (even after over a year) and in tears (up to 200 days) when they were frozen to low temperatures. In spite of these problems, analysis of unconventional biosamples (e.g., meconium, nails and tears) may be advantageous and provide information that would not be accessible in materials routinely analyzed (e.g., urine and blood). Meconium and nails have a wider detection window than the routinely examined body fluids and some unconventional materials (e.g., oral fluid or sweat). Meconium can be used as the material of choice for evaluating fetal exposure to therapeutic drugs and drugs of abuse throughout pregnancy, because this specimen begins accumulating in the fetal bowel some time during 12–16 weeks gestation and continues to accumulate until birth. Thus, analysis of meconium of a newborn infant may serve as a marker of drugs and other xenobiotics exposure, including tobacco smoke, over the last two trimesters. This specimen is of special interest in analyzing non-persistent drugs. An early, correct diagnosis of drug exposure for a baby in uterus

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is the newbornÕs best chance of receiving appropriate treatment. Analysis of nail (fingernail and toenail) plates to determine licit and illicit drugs also presents a particularly useful tool for forensic toxicology. Some studies, especially those concerning detection and determination of illicit drugs (e.g., cocaine, morphine, cannabis and AM-related compounds), and including their many metabolites, confirmed the usefulness of nails as a complement or alternative to hair. Generally, nails may serve as potentially useful biological specimens for detection of past use of narcotic drugs. In some cases, morphine and cocaine concentrations were higher in toenails than hair. However, external contamination of the analyzed nails (especially fingernails) by these drugs needs to be taken into account. Moreover, nails, as they have similar levels of drugs and their metabolites to hair, may provide supporting evidence in forensic cases concerning drugfacilitated sexual assaults. Human-tear samples have been employed for pharmacokinetic study of some antihistamine drugs, and antibiotic and antimicrobial agents, which are administered for treatment of allergic conjunctivitis, ocular infections or skin infections, and respiratory-system and sexually-transmitted diseases. Pharmacokinetics of antimicrobial drugs, used in veterinary cases (e.g., pradofloxacin and doxycycline) have also been investigated, so as to draw appropriate conclusions. Among other drugs, analgesic [e.g., acetaminophen (paracetamol)] and antiepileptic (e.g., valproic acid) drugs have been determined in human tears. The concentrations of the drugs found were compared with the corresponding findings in serum or plasma samples. The chief purpose of determinations of these drugs was to evaluate the potential applicability of tears as an alternative material for therapeutic drug monitoring, and the studies performed showed strong correlations between drug concentrations in blood and tears. The main advantageous feature of the alternative specimens (e.g., meconium, nails and tears) is their noninvasive sampling (meconium) or almost non-invasive sampling (nails, tears). In particular, when meconium is analyzed, no parental consent is necessary to take samples of this specimen. However, to obtain reproducible results, some problems with sampling of tears need to be avoided and some drawbacks of both commonly used collection methods (direct and indirect) need to be taken into account. Considering the characteristics and the analytical potential of these three biological materials, we may anticipate that their application in drug analysis will grow, at least in a few analytical fields:  assessing drug exposure during pregnancy (meconium);  forensic toxicology (nails);

Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

 pharmacokinetic study (tears); and,  clinical drug monitoring (tears). However, more common use of these materials requires further investigations focusing on quantitative analysis of the drugs of interest in these specimens and comparison studies with other biological matrices. References

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