Detection of Stanozolol in Hair by Negative Ion Chemical Ionization ...

ForensicScienceInternational

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84 (1997) 201-209

Forensic Science Internhml

Detection of alprazolam in hair by negative ion chemical ionization mass spectrometry Karin M. Hiild”p*, Dennis J. Croucha, Diana G. Wilkins”, Douglas E. Rollins”, Robert A. Maesb “Centerfor

Human Toxicology Depament of Pharmacology and Toxicology, Universi~ of Utah, 490 Biomedical Polymers Building, SaltLake City, UT 84112, USA bCJtrecht Institute of Pharmaceutical Sciences (UIPS), Utrecht University Department ofAna&sis and Toxicology, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands

Abstract

A sensitive and specific method for the quantitative determination of alprazolam (AL) in hair has been developed. After the addition of deuterium labeled triazolam as an internal standard, hair samples (20 mg) were digested with 1 N NaOH at 40°C overnight. Calibrators containing known concentrations of AL dried onto drug-free hair were also prepared and digested. After digestion, the solution was cooled, adjusted to pH 9 with 6 N HCl and 1 ml of saturated sodium borate buffer was added. The digested solutions were extracted with toluene:methylene chloride (7:3) and the organic phase was evaporated to dryness. Extract residues were treated with BSTFA and 1% TMCS and analyzed on a Finnigan-MATTM mass spectrometer in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200TM capillary column using hydrogen as the carrier gas. The assaywas capable of detecting 25 pg/mg of AL and was linear to 250 pg/mg. Intra-assay precision was 11.1% at 25 pg/mg and 5.4% at 150 pg/mg. Inter-assay precision was 11.2% at 25 pg/mg and 5.3% at 150 pg/mg. This method has been used to study the hair incorporation of AL into Long-Evans rats who received 5.0 mg/kg or 7.5 mg/kg i.p. twice a day for 5 days. Preliminary results indicate that the AL concentration in the pigmented and non-pigmented hair on day 14 ranged from 60 to 100 pg/mg. 0 1997 Elsevier Science Ireland Ltd. All rights reserved Keywords:

Alprazolam; Benzodiazepines; Hair; GC/MS

*Correspondingauthor. e-mail: [email protected] 0379-0738/97/$17.000 1997Elsevier ScienceIreland Ltd. All rights resewed PII

SO379-0738(96)02063-4

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1. Introduction Benzodiazepines (BZPs) are important to toxicologists because of their frequent use and their potential to affect performances such as driving a motor vehicle or flying an aircraft [1,2]. Historically, to determine BZP abuse, screen tests were performed on the urine or other biological specimens followed by confirmation and quantitation with GC/MS [3-S]. In the last decade, the analysis of hair for the detection and quantification of xenobiotics gained widespread attention in the forensic and clinical communities. Analysis of hair provides a means of determining long-term drug use history because drugs and their metabolites are deposited into the hair [9]. Sramek et al. [lo] and Kintz et al. [ll] were the first researchers who studied BZPs in hair. In 1992 Kintz et al. [ll] detected diazepam, desmethyldiazepam, flunitrazepam and nitrazepam in human hair of chronic BZP abusers using an immunoassay and confirmation with GC/MS, followed by the detection of nordiazepam and oxazepam in human hair in 1996 [12]. In another study using RIA, diazepam was detected in human hair specimens obtained from hospitalized psychiatric patients receiving diazepam, alprazolam (AL), and lorazepam as part of their therapy, whereas AL and lorazepam, the newer BZPs were not detected [lo]. These findings were predictable since AL has a shorter half-life than diazepam, smaller doses are required for therapeutic efficacy, and lower plasma concentrations are found. Also RIA is five times more sensitive for diazepam than for AL. These factors result in the requirement for increased analytical sensitivity for the detection of AL. The objective of the present study was to determine if AL is deposited into hair samples from Long-Evans rats receiving 5 mg/kg or 7.5 mg/kg, twice a day, i.p., for 5 days by using a sensitive and specific method, previously developed for the analyses of AL and a-hydroxyalprazolam (OH-AL) in plasma [13]. 2. Material and methods 2.1. Reagents

AL and the internal standard ‘H,-triazolam (TR-d,) were obtained from Radian Corporation (Austin, TX). AL for dosing was obtained from Upjohn Laboratories (Kalamazoo, MI). High-purity-grade toluene and capillary GC/MS grade methylenechloride, ethylacetate and methanol were obtained from Baxter (McGraw Park, IL). Analytical grade sodium borate was obtained form Mallinckrodt (St. Louis, MO). N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% Trimethylchlorosilane (BSTPA + 1% TMCS) was purchased from Pierce Chemical (Rockford, IL). 2.2. Stock solutions and preparation of standard curves

Drug-free rat hair was obtained by shaving the non-pigmented areas of Long-Evans.rats. Drug reference solutions were diluted in methanol to obtain separate working solutions containing 0.01 rig/ml, 0.1 rig/ml and 1.0 rig/ml of AL.

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Stock and working solutions were stored at - 20°C until use. The diluted working solutions were used to prepare daily standard curves by fortifying drug-free hair with known concentrations of standards. To fortify hair, drug-free hair was carefully cut into small segments and 20 mg were placed into silanized glass vials, and the standard solution was added. The final fortified hair standard concentrations of AL were: 0, 5.0, 12.5, 25.0, 37.5, 50.0, 100, 150, and 250 pg/mg, prepared in approximately 20 mg of non-pigmented drug-free hair. 2.3. Preparation of quality control samples

Quality control samples (25 pg/mg and 150 pg/mg in fortified hair) were prepared daily. Stock solutions used to prepare quality control samples were prepared independently from those used to prepare standards. Drug-free hair was also digested, extracted, and analyzed as a negative control in each assay. 2.4. Alprazolam administration

and hair collection

Male Long-Evans rats (90-150 g) were kept in a constant room temperature environment with an alternating 12-h light and dark cycle with food and water available ad lib. Animals were housed individually in hanging wire cages to prevent contamination from the bedding or the urine or saliva of other rats. AL (250 mg), dissolved in 2 ml of 1.0 M HCl and diluted to 2.5 mg/ml or 5.0 mg/ml with sterile saline, was administered i.p. twice per day in a dose of 5 mg/kg (n = 8) or 7.5 mg/kg (n = 4) for 5 days. On day 0 (prior to dosing) the pigmented stripe on the back and an area of both non-pigmented sides of the animal was shaved to the skin using an electric animal shaver (Thomas Scientific, Swedesboro, NJ). The same areas were shaved on day 14 and day 28 after dosing. Drug-free hair was collected prior to dosing from Long-Evans rats in another dosing experiment. Hair was stored at - 20°C until analysis. Hair collected from these animals was not washed prior to analysis. 2.5. Digestion and extraction

Rat hair (lo-25 mg> from days 0, 14 and 28 was carefully cut into small pieces and mixed. Between 10 and 25 mg of hair was used for the analysis of AL dependent on the amount of hair collected on day 0, 14 and 28. Before the digestion, 50 ~1 of TR-d, (5 ng) was added to the hair standards, controls, or rat samples as the internal standard (final concentration 250 pg/mg). The hair was completely solubilized (digested) with 2 ml of 1 N NaOH at 40°C overnight. After digestion, the tubes were cooled in a freezer for 10 min, the pH of the samples was adjusted to 9.0 with 6 N HCl and 1 ml of saturated sodium borate buffer was added. The tubes were mixed and 7 ml of toluene:methylene chloride (7:3) were added. All tubes were capped, rocked gently for 30 min, and centrifuged for 10 min at 2500 rpm. The organic phase was transferred to clean silanized tubes, and the

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solvent was evaporated at less than 40°C under a stream of air. Twenty-five microlitres of ethyl acetate followed by 25 ~1 BSTFA and 1% TMCS were added to the tubes. All tubes were heated at 80°C for at least 30 min. The tubes were removed from the heating block and allowed to cool to room temperature. The liquid was transferred to labeled autosampler vials for injection into the GC/MS. 2.4. GC / MS analysis

Analyses were performed on a Finnigan-MAT 4500 GC/MS equipped with INCOS@ software, and a CTC A200 S autosampler (Finnigan MAT, San Jose, CA). The chromatographic column was a Restek-200TM capillary column (15 m, 0.25 mm i.d., 0.25 pm) (Bellefonte, PA). The initial column temperature of 190°C was held for 1 min, programmed to 320°C at the rate of 20”/min and held at the final temperature for 2 min. The carrier gas was hydrogen. The temperatures of the injection port, interface, transferline and ionizer were 275°C 310°C 300°C and 130°C respectively. The reagent gas was methane with a source pressure of 0.60 Torr. Emission current, electron energy, conversion dynode and electron multiplier were set at 0.15 mA, -70 eV, -3 kV and - 1500 V, respectively. The mass spectrometer was operated in the negative chemical ionization detection mode. One microlitre of reconstituted extract was injected onto the column in splitless mode. The mass spectrometer was set to monitor ion currents at m/z 308 and 310 for AL and TR-d,, respectively. 2.7. Quantitation of hair extracts

The peak height ratio for each standard, control and sample was calculated as the ratio m/z of 308/310. A standard curve for AL was constructed by plotting the peak height ratios of each standard versus the concentration of AL. This procedure was followed by regression analysis of the standard curve by the method of least squares using Cricket Graph software (Malvern, PA). The concentration of the hair samples was calculated from the regression standard curves. 2.8. Stability and recovery of alprazolam

To assessthe stability of AL under the digestion conditions, AL (1 ng/mg of hair) was added to drug-free hair samples. Internal standard was added to the hair before digestion. The hair samples were incubated with either 2 ml of 1 N NaOH at 55°C for 1.5 h (n = 3) or 2 ml of methanol at 21°C for 1.5 h (n = 3). This was followed by adjustment of the pH and extraction as described above. The mean of the peak height ratio of AL to the internal standard was compared to the mean ratio obtained for drug-free hair samples with internal standard (n = 3) treated in the same way but with AL added after digestion and pH adjustment. Recovery studies were performed by preparing two sets of samples (A and B). Set A consisted of samples with n = 10 at two different concentrations: 25 pg/mg and 150 pg/mg; this set was digested and extracted using the method as presented.



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Samples in set B were digested and extracted as described, except AL was added to the organic solvent after transferring (just prior to evaporation). The ratio of analyte height to deuterated internal standard height was obtained, and mean ratios were calculated for samples in sets A and B. The mean ratio at each of the two concentrations for set A was divided by the mean ratio for the corresponding concentration in set B to obtain the recovery ratio. The recovery ratio was multiplied by 100 to calculate the percent recovery. 3. Results and discussion 3.1. Analytical

method

Very few studies have analyzed BZPs in hair and most studies have focused on diazepam, flunitrazepam and nitrazepam [lO,ll]. Previously, Couper et al. [14] found that under alkaline conditions at 70°C a number of BZPs were degraded. Incubation with methanol did not affect the stability of the BZPs. Therefore, an experiment was performed to determine the stability of AL under the conditions of chemical digestion. Mean recoveries after NaOH digestion of 80.6% and 86.7% following methanol digestion were obtained, therefore the loss of AL due to degradation during digestion was negligible. The overall mean recovery after chemical digestion and extraction at low (25 pg/mg) and high (150 pg/mgl concentrations were 99.8% and 92.2% respectively. Fig. 1 contains reconstructed ion chromatograms of extracts of rat hair from prior to dosing (day 0) and 14 days after a dose of 7.5 mg/kg. Fourteen days after dosing the non-pigmented and pigmented hair contain significant amounts of AL. The small peaks (m/z 308) in Fig. 1A and 1B (day 01 have the same retention time as AL. An explanation for these peaks is not found yet. However, these peaks are also observed in point zero of the standard curve and have the same peak height. Treatment with BSTFA + 1% TMCS improved the peak shapes of AL and TR-d,. Theoretically, only hydroxylated metabolites and not AL and TR-d, should form O-TMS derivatives. However, as demonstrated in a previous article [13] we believe that TMS may affect the chromatography of AL and TR-d, by either ‘associating with’ the drug or by de-activating the GC column and, therefore enhancing the chromatography. Peak-height ratios (AL-d,/TR-d,) were calculated for each standard and plotted against the known concentration of the standard. Simple linear regression correlation coefficients of these standard curves were calculated using Cricket Graph software (Malvern, PA). Correlation coefficients (r2) were typically > 0.90 and the assay was linear to 250 pg/mg hair. To enhance quantitative accuracy at low concentrations the five closest standard curve values were used. The limit of quantitation of the assaywas 25 pg/mg. Intra- and interassay precision of the analytical method were determined by analyzing two different concentrations of fortified hair standards in batches. For intra-assay precision, each concentration studied was analyzed in replicates of five. For inter-assay precision, samples were analyzed in duplicate in five batches


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50.9 , 310

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Fig. 1. Reconstructed selected ion chromatograms of extracts of hair 0 and 14 days after dosing: (A) non-pigmented hair day 0, (B) pigmented hair day 0, (C) non-pigmented hair day 14, and (D) pigmented hair day 14.

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analyzed on separate days. The mean measured concentration for each batch was then used to calculate inter-assay precision and accuracy. The mean, standard deviation, and percent coefficient of variation (%CV) were calculated at each concentration for AL. As shown in Table 1, %CV of both intra- and interassay precision experiment was less than 15%. 3.2. Quantitation of alprazolam in rat hair

The described GC/MS assay was utilized to quantitate AL in rat hair at 0, 14 and 28 days after dosing. Hair from day 0 and 28 contained no AL. The mean concentrations of AL in the non-pigmented rat hair collected on day 14 were 61.9 and 90.0 pg/mg for 5.0 mg/kg and 7.5 mg/kg, respectively and in the pigmented hair 71.5 pg/mg and 70.3 pg/mg, respectively (Fig. 2). There was no significant difference between AL concentrations in pigmented or non-pigmented hair, neither between 5.0 mg/kg and 7.5 mg/kg. However, for the non-pigmented hair a trend demonstrated that an increased AL dose resulted in an increase in AL concentration. These results are made more difficult to interpret since the rat hair grows in waves in which the activity of the follicles is synchronized. Also, the rats of the two dosing-groups were not the exact same age and accordingly the growth phases were different. Hair growth is an important factor in drug incorporation. Because the growth phases of the two dosing-groups may have been different the rat hair may not have incorporated the same amount of drug. Therefore, the results of the 5.0 mg/kg and the 7.5 mg/kg dose can not be simply compared. In an experiment with Sprague-Dawley rats of the same age, it was demonstrated that higher doses of codeine resulted in higher concentrations of codeine and its metabolite morphine in rat hair [151. To prove that the incorporation of AL into rat hair occurs in a distinct dose-dependent manner, AL concentrations in rat hair that is in the same growth-phase must be compared.

Table 1 Inter- and intra-assay accuracy and precision for the analysis of hair fortified Target concentration Wd Intra-assay 25 150 Inter-assay 25 150 a % CV = Percent coefficient

with AL

Accuracy (L7cof target)

Precision (o/o CV)”

110.4 107.9

11.1 5.4

101.6 101.4

11.2 5.3

of variation.

KM HGld et al. /Forensic


80 0

60

NP

-P

40 20 i

0 5

mg/kg

7.5

mg/kg

Fig. 2. Mean hair concentrations on day 14 of AL from rats receiving 5 mg/kg or 7.5 mg/kg twice a day, i.p., for 5 days. The hair on day 0 and 28 was blank. The mean concentrations of AL in the non-pigmented (NP) rat hair collected on day 14 were 61.9 pg/mg and 90.0 pg/mg for 5.0 mg/kg and 7.5 mg/kg, respectively and in the pigmented hair 71.5 pg/mg and 70.3 pg/mg, respectively.

4. Conclusion

This paper describes a negative chemical ionization GC/MS procedure for the quantitative analysis of AL in rat hair. The method was sensitive, precise and accurate. AL was easily detected in both non-pigmented and pigmented rat hair 14 days after dosing at levels in the 60-100 pg/mg range. Studies are currently performed to investigate the possibilities of detecting the new generation BZPs, such as AL, in human hair. Acknowledgements

This research was supported by NIDA Grant Nos. DA09096 and DA07820. The authors wish to thank Mr. A.C. Spanbauer for his advice and assistance. References [l]

[2] [3]

[4]

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191 K.M. Hold, Drug testing in hair. AACC TDM/

[lOI illI [121 1131

[141 [151