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Chemicals, preparation of internal and calibration standards, Instrumentation, Method Validation, and Nyaope sample profiling and comparison

CRISPRAnalytical Chemistry nyaope mass selective detector (Agilent 5975 CVL MSD) Agilent Technologies system (Chemetrix, RSA) fusedsilica capillary column HP-5MS HeroinCannabisWarfarinDrug AnalysisGas Chromatography–Mass Spectrometry


Tertiary butyl alcohol (t-BuOH) was purchased from Merck (Darmstadt, Germany), tetracosane 99% was purchased from Sigma-Aldrich (St Louis, MO, USA), isopropanol (i-PrOH)-AR grade was purchased from Associated Chemical Enterprise (Johannesburg, South Africa). Representative compounds, identified in casework samples of nyaope by the South African Police Service (SAPS), were used to validate the GC– MS method. Certified reference standards of Δ9 -tetrahydrocannabinol (Δ9 -THC) and diamorphine (both 1 mg/mL) were purchased from Cerilliant-Sigma Aldrich (Austin, TX, USA). Caffeine and phenacetin were purchased from the US-Pharmacopeia (Rockville, MD, USA) as USP powder reference standards while efavirenz and nevirapine were purchased from WHO International Chemical Reference Substances (Strasbourg, France) as ICRS powder reference substances.

Preparation of internal standards

The internal standard solution, tetracosane (C24), was prepared at a final concentration of 0.02 mg/mL in tertiary butyl alcohol. Tertiary butyl alcohol has previously been shown to be the solvent of choice for presenting nyaope extracts to the GC–MS.12 The internal standard solution was used to dilute the certified reference standards, and other samples, before GC–MS analysis.

Preparation of calibration standards

Stock solutions (1 mL at 1 mg/mL) of Δ9 -THC in methanol and diamorphine in acetonitrile were placed in an amber GC–MS vial, evaporated to dryness under nitrogen and then re-dissolved in 1 mL of the internal standard solution to give stock solutions of 1 mg/mL. Phenacetin, caffeine, efavirenz and nevirapine were dissolved at concentrations of 1.03, 1.00, 0.998 and 1.05 mg/mL, respectively, in the internal standard solution. From these, 14 standards in the concentration range 0–1.0 mg/mL, at notional concentrations of 0, 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75 and 1.0 mg/mL were prepared.


GC–MS analysis was carried out using an Agilent Technologies system (Chemetrix, RSA) consisting of a gas chromatograph, Agilent 7890A, and mass selective detector (Agilent 5975 CVL MSD) with an auto sampler 7683 B series (1 µL injection). Chromatographic separation was performed using a computer-controlled autosampler with a fusedsilica capillary column HP-5MS (30 m x 0.25 mm i.d., film thickness 0.25 µm; J&W Scientific, Folsom, CA, USA). Splitless injection was used at 280 °C. The GC oven temperature programme consisted of an initial temperature of 100 °C for 0.4 min, raised to 290 °C at a rate of 60 °C/min, held at 290 °C for 2.4 min then raised to reach 316 °C at 60 °C/min and held for 3 min. The total run time was 9.4 min. High-purity helium (99.9995%) was used as the carrier gas, at a flow rate of 1 mL/ min. The MS parameters used were as follows: the interface temperature was 280 °C, the inlet temperature 250 °C, the ion-source temperature 230 °C, electron ionisation was achieved with a source voltage of 70 eV and the mass spectrometer (quadrupole) was used in scan mode. The spectra were recorded in the scan range (m/z) 35 to 550 amu, at a scan rate of 1 scan/s.

Method Validation

The method was validated by determining the precision of the retention index of each compound, the linearity of detector response, the limit of detection and of quantitation, repeatability and the reproducibility of the measurements.13,14 The precision of the retention index was obtained for each compound (phenacetin, caffeine, efavirenz, nevirapine, Δ9 -THC and diamorphine) by calculating the mean, standard deviation and relative standard deviation of the retention index, relative to tetracosane, for 10 replicate analyses. Linearity of the detector response to the exemplar drugs was determined by preparation of calibration curves for samples in the concentration range 0.00–1.00 mg/mL. The regression equations for detector response relative to the internal standard, the value of R2 and residual plot analysis were used to confirm linearity of detector response. Limit of detection (LOD) and limit of quantitation (LOQ) were determined by the calibration curve slope using reference sample solutions with concentrations in the vicinity of the LOD15, namely 0.000, 0.001, 0.0025, 0.005, 0.01 and 0.05 mg/mL and the equations: LOD = s 3.3 x σ x CIS Equation 1 LOQ = s 10 x σ x CIS Equation 2 where σ=standard error of the measured response, S=slope of the regression line and CIS=concentration of the internal standard = 0.018 mg/mL To measure the accuracy of the method (closeness to true concentration), 10 replicate measurements of standards of known concentrations were made, the experimental concentrations determined and Equation 3 applied: % Accuracy = x 100 Measured concentration actual concentration Equation 3 Precision is a measure of the closeness of the analytical results obtained from a series of replicate measurements of the same measure under the conditions of the method. Intra-assay precision (repeatability) and inter-assay precision (reproducibility) were assessed using drug standard mixtures of phenacetin, caffeine, efavirenz, nevirapine, Δ9 - THC and diamorphine at three concentration levels (0.01, 0.1 and 1.00 mg/mL). Repeatability was assessed by making 10 replicate analyses of the drug standards at three concentration levels and calculating the mean, standard deviation and relative standard deviation of the relative response to the internal standard. Reproducibility was assessed by making five replicate analyses of the drug standards over five consecutive days at the three concentration levels, and calculating both within group (W) and between group (B) precision using one-way ANOVA (Group = Day)16: %RSDw = MSW x x 100 Equation 4 %RSDB = (MSB - MSW)/n x x 100 Equation 5 If MSB < MSW, set %RSDB = 0 3 Volume 117| Number 11/12 November/December 2021 Research Article where RSD = relative standard deviation, X = grand mean of all observations, n = number of observations in group, MSW = mean of squares within group, MSB = mean of squares between groups.

Nyaope sample profiling and comparison

In order to investigate the validity of the method for nyaope sample identification and comparison, both simulated samples and casework samples of nyaope were analysed. Street cannabis and heroin samples seized by the SAPS were used to prepare simulated nyaope samples. Three blind simulated samples were prepared by mixing a heroin street sample, a cannabis street sample, efavirenz tablets and nevirapine tablets, in different combinations and proportions, to mimic as closely as possible a typical street nyaope sample. The three mixtures were homogenised by grinding using a mortar and pestle. The samples were then further divided into six blind sub-samples each to give a total of 18 samples marked S1–S18. Homogenised samples which had a mass ranging between 12 mg and 14 mg were mixed with 3 mL of the internal standard solution (0.02 mg/ mL tetracosane in tertiary butyl alcohol) in a 20-mL head space vial. The mixture was sonicated for 15 min12,17,18, filtered into amber GC–MS vials and analysed in triplicate. Each of the extracts of the simulated samples S1–S18 was analysed at 0, 24, 48 and 72 h in order to confirm the stability of the extract once prepared.13 Additionally, chromatograms of members of each of the three groups were compared at the same time intervals to determine whether samples from the same parent batch could be discriminated after these time intervals. Finally, chromatograms from one member of each of the three groups were compared at these time intervals to demonstrate whether it is possible to discriminate between groups. Five casework samples of nyaope were ground into a fine powder using a mortar and pestle. Sub-samples (circa 12–14 mg) of these street samples were placed in a 20-mL vial and extracted with 3 mL of the internal standard solution prior to analysis. Each of the casework samples was analysed in triplicate by GC–MS at t=0 after extraction. The chromatograms were compared to determine whether it was possible to discriminate between street samples. Each extract was then analysed after 24, 48 and 72 h to demonstrate stability of extracts of such samples. Semi-quantitation was conducted on caffeine, diamorphine and Δ9 -THC for each of the five casework samples. Two unsupervised chemometric methods – agglomerative hierarchical cluster analysis (HCA) and principal component analysis (PCA) – were performed on both the blind simulated and casework nyaope samples using the XLSTAT statistical and data analysis solution 2019 version. The HCA and PCA analysis were conducted for the samples analysed at 0, 24, 48 and 72 h.

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