Chemicals and reagents
Dichloromethane (DCM), acetone, methyl tertiary butyl ether (MTBE), formic acid, and sodium hydroxide were all provided by the Department of Chemistry, Author University, Lahore, Pakistan, whereas Certified Reference Material (CRM) for internal standard deuterated 6-MAM, of purity ≥ 99%, was bought from Cayman Chemical (MI, USA).
Sample collection
After taking prior consent, 10–30 hair strands were taken from the scalps of each of the 20 random heroin addicts from a well-known area of Darbar Road at Lahore, Pakistan. A further 20 samples of subjects were taken, with prior consent, from Genius Rehab center situated at Barki Road, Lahore (Pakistan). The study approval was given by the Department of Chemistry and Life Sciences, Government College University, Lahore, with approval letter number, GCU/1-056/CLS. The participants were included after approval from the “Office of Research, Innovation and Commercialization” (ORIC) with reference number 2017-0057. All the participants were informed about the study protocol and then included after taking verbal consent. The inclusion criterion set for subjects at the rehabilitation center was those who have gone through at least 30 days of rehabilitation period against heroin abuse. The average duration of a patient at the rehabilitation center for sample collection, in our study, was 35 days. The hair samples from all participants were taken from the apex portion of the head, in order to avoid ambiguity in the dispersal of 6-MAM in hair due to inconstant rate of hair growth in different parts of the head (Tables 3 and 4).
Sample inclusion criteria
a) Only those individuals who had been admitted to the mentioned de-addiction center at least 30 days prior to the sample collection date were selected.
b) Healthy other than heroin addiction subjects of different age groups were included for this study.
Sample exclusion criteria
a) Individuals who got admitted to the mentioned de-addiction center against heroin abuse less than 30 days at the sample collection date were omitted from this study.
b) Individuals who had not taken heroin for at least 3 days (for addicts) prior to the date of sample collection were not considered for this study.
c) Individuals presenting any psychotic disorder or any other physical complaint or comorbid situation were excluded from the study.
d) Individuals who had experienced any kind of surgery, cosmetic hair treatment, or bald were excluded from this study.
Sample analysis
Each round of hair analysis comprised of the following steps:
a) Sample decontamination
b) Digestion and extraction of the hair sample
c) Quantification of 6-MAM
Sample decontamination
Approximately, 50 mg of each sample was washed at room temperature, firstly with distilled water for 5 min, then with acetone for 1 min, and finally with dichloromethane for 2 min. The sample was air dried and then cut into small segments of about 3–4 mm [15].
Digestion and extraction of the hair sample
The washed samples were incubated with 500 μL of 1 M NaOH for 3 h in a water bath at 50 °C to allow for digestion. Samples were then extracted with 2 mL MTBE for 30-min rotary mixing and for 10-min centrifugation. Solvent layers were then shifted into clean tubes containing 150 μL 1% formic acid, then mixed on a rotary mixer for 20 min, and were removed after 10 min of centrifugation by aspiration. From the remaining aqueous layer, a 1-μL sample was injected into the GC–MS system [16,17,18].
Quantification of the 6-MAM
Shimadzu’s GC–MS-QP-2010 was used for the quantification of 6-MAM. The column was HP-5 crosslinked 5% phenylmethyl polysiloxane fused-silica capillary column (25 m (length) × 0.32 mm (internal diameter) × 0.17 μm (film thickness)). Helium of purity 99.9% was used as carrier gas. The temperature of the injector was 280 °C and the flow rate of the carrier gas was 1 mL/min. The temperature of the oven was maintained at 110 °C for 3 min at 10 °C/min, followed by 210 °C for 2 min at 10 °C/min, and finally at 300 °C for 5 min at 20 °C/min. SIM mode was used for quantitative analysis of 6-MAM (m/z <300) [19].
Method validation
The method was validated in terms of selectivity, linearity, LOD, LLOQ, carry-over, precision and accuracy, recovery, and stability according to the protocols established previously [20, 21].
To evaluate selectivity, blank hair samples obtained from five different origins were analyzed to determine endogenous compounds or potential interferences released from the matrix. LOD and LLOQ were measured by evaluating the signal/noise (S/N) ratio of 10 replicates of blank hair for each compound at proper concentrations. LOD was calculated on the basis of the concentration with a S/N > 3, while the concentrations of the target compounds with a S/N > 10 were chosen as LLOQ. The absence of carry-over was evaluated by injecting at the highest point of the calibration curve, followed by a solvent blank, and measuring the peak area at the retention times of the target compounds under investigation. For routine analysis of hair samples, ethyl acetate blanks were run between each pair of samples. The linearity of the method was evaluated over the concentration ranges of 0.1–25 ng/mg for the sample in both groups and was expressed by the determination coefficient (R2). The calibration curves were obtained by least-squares linear regression. The intra-day precision and accuracy of the method were established by eight independent determinations of the samples (n=8). The inter-day precision and accuracy were determined in two different days for the aforementioned replicates over a 5-day gap for each replicate. To determine the precision, the coefficients of variations (CV%) were calculated for the replicate measurements. Accuracy (bias%) was expressed as the relative error of the calculated concentrations and was calculated by the degree of agreement between the measured and the nominal concentrations of the QC samples. For recovery determination, QC samples were prepared at three concentration levels. The recovery was determined by comparing the absolute peak area (A) of the compound for the QC samples prepared in three replicates with the absolute peak area (B) of the target compound for the samples processed as blank and spiked at the same concentration level. The recovery was calculated using the following equation: Recovery (%) = A/B × 100. Sample stability was assessed by repeated analysis of low-concentration QC and high-concentration QC samples (n = 3), spiked at mentioned concentrations. To examine the stability, QC hair samples were left for 10 days prior to sample preparation and analysis. The internal standard used was a deuterated 6-MAM. These samples were analyzed, and the peak area ratios were compared with the ones obtained by the analysis of freshly prepared samples.