We analysed four different Sudanese roselle samples for their potential as novel xanthine oxidase (XO) inhibitors. Phytochemical screening showed the presence of polyphenols, flavonoids, organic acids, saponins and sterols in all samples. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to identify and characterise five anthocyanins in all samples: cyanidin-3-glucoside (cy-3-glu), delphinidin-3-sambubioside (dp-3-sam), cyanidin-3-rhamnoside (cy-3-rhm), delphinidin-3-rhamnoside (dp-3-rhm) and pelargonidin-3-glucoside (pg-3-glu). Identification of cy-3-rhm, dp-3-rhm and pg-3-glu confirmed the selectivity and sensitivity of LC-MS as a powerful technique for identifying anthocyanins. In silico studies of the identified anthocyanins were performed to explore their promising inhibitory activity toward XO. Interactions between the ligand and the enzyme were via the H-bond, and hydrophobic (π-alkyl, π-sigma and alkyl) and/or electrostatic (π-cation) bonds. Inhibition of the anthocyanins was compared with that of topiroxostat, a commercial drug for hyperuricaemia. Dp-3-rhm was the most active inhibitor with a binding energy of ca. -10.90 kcal/mol compared to topiroxostat’s binding energy of ca. -8.60 kcal/mol. “The good inhibition results obtained from anthocyanins toward XO suggest their application as a drug candidate to treat gout and other diseases related to the activity of XO.

Phytochemicals and in silico investigations of Sudanese roselle

**_Chemicals_**

All chemicals were of analytical grade except those used in LC-MS analysis which were HPLC-grade.”

_**Phytochemical screening**_

**Preparation of extracts**

The extract was prepared according to Wrolstad et al.’s18 alternative protocol with some modifications in the purification step. The purification was established by simple liquid-liquid extraction using an organic non-polar solvent (petroleum ether) and (0.01% (v/v) HCl) acidified methanol to remove water-insoluble impurities. The desired layer was reserved, the solvent was evaporated using a rotatory evaporator (Heidolph Laborota 4000 efficient HB digital, Germany) and allowed to air dry for 3–4 h. The attained gummy extract was used for all the phytochemical qualitative and quantitative analyses after dissolving in 1–2 cm3 methanol for each qualitative test.

**Preliminary screening**

Qualitative analysis was carried out according to methods described by Harborne10 and Sofowara19 via simple test tube tests. These are the test for polyphenols (FeCl3 test), test for flavonoids (AlCl3 test), test for sterols (H2SO4 test), test for saponins (Frothing test), and test for anthraquinones (KOH test).

**Thin-layer chromatography**

An amount of 10 mg from the gummy powdered extract of each of the samples was dissolved in 1 cm3 of methanol and, using a capillary tube, spots were made from each sample extract on an aluminium sheet of pre-coated silica gel thin-layer chromatography plates, then elution was carried out in a covered thin-layer chromatography chamber using the mobile phase of a mixture of chloroform and methanol (7:3 v/v) according to Harborne10. The procedure was repeated on two other plates.

After performing the elution, plates were examined immediately in daylight, then sprayed with chemical reagents and visualised under a UV lamp (at 365 nm). Bromocresol green was the spray reagent employed for detection of organic acids, while AlCl3 was used for detection of flavonoids.20

**Quantitative analysis**

Total phenolic content was measured using Folin–Ciocalteu’s reagent as described by Wolfe et al.21 Total flavonoid and total anthocyanin contents were measured according to Shanmukha et al.22 and Pacôme et al.6, respectively.

For total phenolic and flavonoid contents, absorbance was measured at 765 nm and 510 nm, respectively, against a reagent blank using a Jenway 7205 UV/Vis double-beam spectrophotometer. The concentrations of phenolics and flavonoids in the test sample were determined and expressed as gram equivalent of gallic acid (g GAE) and quercetin per gram of air-dried extract (g ADE), using a standard calibration plot for each individually. All measures were performed in duplicate.

Determination of total anthocyanins

Total anthocyanin content was measured at an absorbance of 530 nm using a Jenway 7205 UV/Vis spectrophotometer against the blank; total anthocyanin content was estimated as cyanidin-3-glucoside at 530 nm using a molar extinction coefficient of 26 900 L/mol/cm) and molar mass (449 g/mol) and was expressed as cyanidin-3-glucoside (g cy-3-glu)/(100 g of air-dried extract).

**LC-MS/MS identification**

Anthocyanin extraction was carried out using 250 cm3 acidified methanol by Soxhlet apparatus2 at a temperature of 50–55 °C. The extract was purified by simple liquid/liquid extraction after reducing the extract volume using the rotatory evaporator (Heidolph Laborota 4000 efficient HB digital, Germany).

Anthocyanins were identified in the purified sample extracts using LC-MS/MS with a UPLC-Q-TOF-MS (XEVO-G2 QTOF YCA119, Waters, using Acquity UPLC HS T3 (100 × 2.1 mm, 1.8 μm; Waters) column maintained at 45 °C in a column oven in the LC-MS Laboratory, Beijing University of Chemical Technology BUCT, China. The analysis was carried out according to the method described by Cahlíková et al.23 with some modifications. The conditions of the chromatographic run were as follows: 0–1 min: 90% A and 10% B, 1–5 min: 85% A and 15% B, 5–10 min: 40% A and 60% B, 10–13 min: 0% A and 100%B, 13–15 min: 90% A and 10% B. Gradient mobile phase composed of water acidified with 0.1% formic acid (solvent A) and acetonitrile (solvent B). The flow rate and sample injection volume were 0.4 cm3/min and 3.00 µL, respectively.

The ion source of the quadropole time-of-flight (Q-TOF) detector was set at the positive electrospray ionisation polarity mode. The optimum conditions of the Q-TOF system, performed in the resolution mode, were: capillary voltage: +3.000 kV, ion source temperature: 100 °C, extractor: ٤.٠ V; RF lens: ٠.٣ V, mass range m/z: 50 to 2000 Da, nitrogen as the desolvation gas at a flow rate of 800 L/h and at a temperature of 400 °C. Nitrogen was used also as the cone gas (٢٠ L/h), and argon as the collision gas. The cone voltage, collision energy, and well time were carefully optimised for each compound and transition individually (cone voltage set at 4/30 V, collision energy set finally at 6 eV). The software used for the MS control and data gathering was Mass Lynx 4.1.

**Molecular docking**

Molecular docking is a method used to screen for the ability of specific small compounds (ligands) to fit into a previously identified therapeutic target (the receptor; usually a protein enzyme).24,25 Several docking programs can be used: AutoDock Vina, GOLD, and MOE-Dock are top ranked with the best scores.25 We used the AutoDock Vina program26 for the docking of anthocyanin ligands to the active site of the XO enzyme, after performing the required preparations.

First, the protein crystal structure and ligand 3D structures were downloaded from RCSB Protein Data Bank27 (1FIQ PDB code) and PubChem online databases28, respectively. Then the suitable binding site of the protein was obtained using Chimera software29 and the ligands were converted via Open Babel software30 from SDF format to pdbqt. Thereafter, using autodock tools31, all water molecules in the protein pdb file were removed, hydrogen atoms were added, and the grid box dimensions were set, then the file saved as pdbqt. Ligands were prepared by detecting torsions for each structure and saved as a pdbqt file. The AutoDock Vina order was then created after preparing the configuration files. The process was repeated for each docking trial for all ligands.

**Data analysis**

The obtained (out.pdbqt) file for each ligand was visualised using discovery studio32 and pymol33 visualisation programs.

Chemicals & Phytochemical Screening (Thin-layer Chromatography, Molecular Docking, LC-MS/MS identification)

Excipients are  the various ingredients, apart from the active pharmaceutical ingredients,   which are added to pharmaceutical formulations. Excipients obtained from   natural sources are preferred over those from synthetic sources because they   are cheap, biocompatible and readily available. Gums are made up of   carbohydrate units which are linked by glycosidic bonds. This study was aimed   at evaluating the potential binding and disintegrating properties of gum   obtained from the bark of Cinnamomum zeylanicum, which was obtained from   Effiduase in the Ashanti region of Ghana. The gum was extracted using 96%   ethanol and the moisture content, Fourier transform infrared spectroscopy   spectra, water holding capacity, swelling index and flow properties of the   gum were determined. The gum was used to formulate tablets at different   concentrations (10% w/ v , 15% w/ v and 20% w/ v ) as binder with acacia as   the standard. The gum was also used to formulate tablets at different   concentrations (5% w/ v , 7.5% w/ v and 10% w/ v ) as disintegrant with   starch as the standard. Quality control tests were then conducted on all   formulated tablets. The gum exhibited good flow and physicochemical   properties. All formulated tablets passed the uniformity of weight test,   friability test, disintegration test, hardness test, uniformity of dimensions   test and drug content. All batches of tablets, except Batch 7, passed the   dissolution test. Based on the study carried out, C. zeylanicum gum can be   used as an alternative excipient to acacia and starch as a binder and a   disintegrant, respectively.

Evaluation of the binding and disintegrating properties of gum obtained from the stem bark of Cinnamomum zeylanicum

**Materials**

The bark of C. zeylanicum was obtained from   Effiduase in the Ashanti region, Ghana, and authenticated at the Department   of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame   Nkrumah University of Science and Technology (Kumasi, Ghana) by a botanist.   It was given a specimen voucher number of KNUST/ HM1/2018/SB002. Diethyl   ether, ethanol (96%), paracetamol powder, acacia powder, starch powder, talc,   magnesium stearate and lactose were obtained from UK Chemicals, Kumasi. All   other reagents used were of analytical grade.

**Extraction of gum from Cinnamomum zeylanicum bark**

Distilled water (7 L) was added to 200 g C. zeylanicum powdered bark and   allowed to stand for 24 h at room temperature (27 °C). The mixture was then   boiled for 15 min, allowed to cool and filtered with a calico strainer to   remove any debris to obtain the gum. Ethanol (96%) was used in purifying the   gum obtained via precipitation. The precipitated gum was filtered, washed   with diethyl ether and dried in a hot air oven at 60 °C for 12 h.8 The dried   gum was milled, passed through a sieve with an aperture size 180 µm and   appropriately stored for use.

**Determination of moisture content**

A mass of 1 g of the gum was transferred into a dried crucible of known weight and placed in an oven at 105 °C for 5 h. The crucible containing the gum was removed and allowed to cool at room temperature and weighed.

**Swelling index and water holding capacity of Cinnamomum zeylanicum gum**

An amount of 0.5 g C. zeylanicum gum (CZG) was transferred into a measuring cylinder, tapped and its volume recorded as V0 . It was then dispersed in 10 mL of distilled water and allowed to stand for 24 h. The volume occupied by the gum was observed after 24 h (V24) and calculated as: Swelling index = V24 - V0 V0 x 100 Contents in the measuring cylinder used for determination of the swelling index were also used to determine the water holding capacity as described by Ofori-Kwakye’s group.

**Determination of the pH of CZG**

Cinnamomum zeylanicum gum (0.1 g) was dispersed in 10 mL of water and stirred for complete dissolution. The pH was then checked with a pH meter (MW101, Milwaukee Instruments, Rocky Mount, NC, USA).11 This was done in triplicate.

**Determination of moisture content**

A mass of 1 g of the gum was transferred into a dried crucible of known weight and placed in an oven at 105 °C for 5 h. The crucible containing the gum was removed and allowed to cool at room temperature and weighed.

**Swelling index and water holding capacity of Cinnamomum zeylanicum gum**

An amount of 0.5 g C. zeylanicum gum (CZG) was transferred into a measuring cylinder, tapped and its volume recorded as V0 . It was then dispersed in 10 mL of distilled water and allowed to stand for 24 h. The volume occupied by the gum was observed after 24 h (V24) and calculated as: Swelling index = V24 - V0 V0 x 100 Contents in the measuring cylinder used for determination of the swelling index were also used to determine the water holding capacity as described by Ofori-Kwakye’s group.

**Determination of the pH of CZG**

Cinnamomum zeylanicum gum (0.1 g) was dispersed in 10 mL of water and stirred for complete dissolution. The pH was then checked with a pH meter (MW101, Milwaukee Instruments, Rocky Mount, NC, USA).11 This was done in triplicate.

**Compatibility studies on formulated granules (Paracetemol powder, CZG)**

Paracetamol powder, CZG and formulated granules were individually scanned using a Fourier transform infrared spectrophotometer (Bruker Alpha II, Germany) over 500–3500 cm-1 wavelengths. Their spectra were then superimposed to assess the presence or absence of principal bands of paracetamol in the formulated tablets.

**Determination of the flow property of CZG**

Cinnamomum zeylanicum gum (30 g) was weighed (M) and transferred into a 100-mL measuring cylinder and the initial volume (V0) recorded. The measuring cylinder was tapped on a bench until a constant volume (Vf ) of the gum was obtained. The bulk density, tapped density, Carr’s index and Hausner ratio were calculated as: Bulk density = M V0 Tapped density = M Vf Carr’s index = (Tapped density - bulk density) Tapped density x 100 Hausner ratio = Tapped density Bulk density The fixed height method was also used in the angle of repose determination. The gum was allowed to freely flow through a clamped funnel to form a heap. The height (h) and diameter (d) of the heap were determined. Using the equation below, the angle of repose (α) was calculated: tan (α) = h/(0.5 d)

**Formulation of tablets**

The wet method of granulation and the technique of doubling the bulk were employed in preparing 12 batches of paracetamol granules. CZG was used as a binder (10% w/v, 15% w/v and 20% w/v) for Batches 1–3 (B1–B3) and as a disintegrant (5% w/w, 7.5% w/w and 10% w/w) for B7–B9. Acacia gum and starch were used as a standard binder and disintegrant, respectively, at the same concentrations stated above for B4–B6 and B10–B12, respectively. All ingredients except talc and magnesium stearate were accurately weighed and mixed together with water as the granulating fluid to form a damp mass. This was then screened with a mesh (2360 µm) and dried at 60 °C for 2 h in an oven. The dried granules were screened with a 1180 µm sieve, mixed with magnesium stearate and talc and compressed into tablets using a single punch tableting machine (TDP 5, Herun, China). Sixty (60) tablets were compressed from each batch to cater for all quality control tests. The constituents of the formulated tablets are given in Table 1.

**Evaluation of tablets Weight uniformity test**

Twenty tablets were randomly selected from each batch and individually weighed to calculate average weight. The average weight and percentage deviation of each tablet were determined as described in the British Pharmacopoeia.14

**Evaluation of tablets Friability test**

Ten tablets from each batch were randomly selected and weighed. They were then placed in a friabilator (EF-2W, Roche, Switzerland) regulated at 4 min at a speed of 25 rpm (revolutions per minute). Afterwards, the tablets were removed from the friabilator, dedusted and reweighed.15 The percentage friability was calculated as: Friability (%) = W0 - W W0 x100 where W is the final weight and Wo is initial weight.

**Disintegration test**

Six tablets from a batch were selected at random and each tablet placed in a tube of the basket-rack assembly in an Erweka disintegration apparatus (ZT 320 series, Germany). The basket rack was placed in a water bath regulated thermostatically at 37±2 °C and observed until all the tablets had disintegrated completely.12 This procedure was repeated for all batches.

**Uniformity of dimension and hardness test of tablets**

Ten tablets were randomly selected from each batch. Their diameters and thickness were determined using vernier calipers. The hardness of ten randomly selected tablets from each batch was determined with a Veego hardness tester (DIGITAB-SPV, India). The tensile strength was calculated as: Tensile strength= 2F πDt where D is the diameter, t is the tablet thickness and F is the diametrical break force of the tablet. Uniformity of drug content Ten tablets from each batch were randomly selected. Each tablet was crushed, dissolved in 50 mL of HCL (0.1 M) and the mixture topped up to 100 mL using 0.1 M HCL. The mixture was filtered and its absorbance determined at 245 nm using a UV spectrophotometer (DU-8800R, Drawell, Shanghai). A standard calibration curve was used to calculate the average drug content for three determinations.

**In vitro dissolution studies**

In vitro drug release of the tablets was determined using the USP II dissolution apparatus (DT6, Erweka, Germany) at 50 revolutions per minute. Six (6) tablets each for all batches were evaluated. A tablet was put in each vessel containing 900 mL of the dissolution medium, HCL (0.1 M), at 37±0.5 °C. A volume of the medium (10 mL) was drawn at time intervals (5, 15, 30, 45 and 60 min) and filtered using Whatman filter paper number 5 with a pore size of 2.5 μm. The volumes taken were replaced to maintain sink conditions. A UV-visible spectrophotometer (Alpha II, Bruker, Germany) was used to check the absorbance of the filtrates at a wavelength of 245 nm. The average drug release profile for three determinations was carried out using a standard calibration curve.16 The various batches of tablets which passed the dissolution profile were analysed for their similarity factor (f2) using the equation: Similarity factor (f2) = 50xlog {1+ (1/n) ∑t=1n (Rt −Tt )2 }-0.5 x 100 where n = dissolution time points, Rt = dissolution value for reference at time t, and Tt = dissolution value at time t.

Materials and methods

Nyaope, a Tswana   word for a mixture or ‘mish-mash’, describes a drug cocktail consisting of   heroin, cannabis, and on occasion other controlled substances and warfarin.   It is highly addictive with extremely unpleasant side effects caused by   withdrawal from the drug. It is a problem drug especially in townships in   South Africa. However, its prevalence in neighbouring southern African states   and further afield is not yet known. There is currently no validated method   for the analysis and comparison of nyaope. We describe a validated method for   the gas chromatography – mass spectrometry analysis of nyaope so that   within-batch and between-batch comparisons of nyaope can successfully be made   for the first time. The validated method managed an accuracy within the range   80–120%, the precision was less than 20% for all analytes and managed   linearity with R2 ≥0.99. The detection limits for diamorphine, efavirenz,   nevirapine and Δ9 -tetrahydrocannabinol were 14.2, 18.6, 18.7 and 9.94 pg on   column, respectively, and the limits of quantitation were 43.1, 56.3, 56.6   and 30.1 pg on column, respectively. The simulated and casework samples were   successfully discriminated into original batches using the identified nyaope   components, the unsupervised chemometric methods principal component analysis   and hierarchical clustering, as well as chromatographic profiles.

A validated method for the analysis and profiling of ‘nyaope’ using gas chromatography – mass spectrometry

**Chemicals**

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.

**Instrumentation**

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 https://doi.org/10.17159/sajs.2021/8738 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.

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

For synthesis of chloro-oximes that can be further reacted to form isoxazoles (Huisgen cycloaddition reaction). 

Reacted oxime to chloro-oxime - formed product, but impurity formed upon leaving the flask out at room temperature overnight. The product turned from yellow to red. 

TCL analysis (1:1 Hexanes:DCM) determined that there was an impurity at baseline. The chloro-oxime was stable enough to remove said impurity by column chromatography to obtain the pure product. 

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