African Journal of & Studies, 14(1), 2015 Copyright © 2015, CRISA Publications iDentifYing the coMPoSition of Street Drug NYAOPE usINg TwO Different MASS SPectroMeter MethoDS

aye a. khine1, kebogile E. Mokwena2, Mempedi Huma2, lucy Fernandes2

1Department of Chemical Pathology, Medunsa/University of Limpopo, 2Department of Public Health, Medunsa/University of Limpopo, South Africa

ABStrAct

Criminalizaton of trading and using of street drug Nyaope has had challenges in South Africa due to controversies about its compositon. The high cost and complexity of its analysis using conventonal chromatography methods also limit the testng availability in most routne laboratories. A state of the Art method with simple specimen processing and faster turnaround tme at an afordable cost is urgently needed. To compare the ability of a new Time-of-Flight Mass Spectrometry with direct sample analysis (TOF-DSA MS) and Gas Chromatography Mass Spectrometry (GC-MS) methods in detectng the consttuents of Nyaope against turnaround tme and cost, in order to recommend a beter system for routne use. Cross-sectonal, qualitatve and descriptve pilot study on samples purchased from various sources of 12 townships in Northern Gauteng Province. The consttuents consistently detected in all samples were cafeine, of abuse such as opiates, , , methyl-dioxy (MDA) and . Some samples contained antbiotcs (citrofex) and antretroviral drugs (zidovudine). Central nervous system (CNS) such as phenobarbitone and , benzitramide, moramide intermediates and thiofentanyl and stmulants such as Pipradol, and fenethyline were detected by the TOF-MS system. The usefulness of TOF-DSA MS was beter as a screening method while GC-MS provides specifcity and confrmatory detecton. Due to direct sample analysis, the TOF-DSA provides analytcal runtme of 15 sec while GC-MS takes 10 minutes per sample. The running cost for the GCMS is more expensive due to the high cost of reference materials and the need to perform specimen preparaton as opposed to TOF-MS. We recommend TOF-DSA MS for inital screening of organic compounds in the Nyaope mixtures followed by confrmaton by GC-MS for medico-legal interventons.

Key words: Nyaope, drug of abuse, Mass Spectrometry, Gas Chromatography, Ant- retrovirals

Corresponding author: Dr AA Khine, MBChB, MMed (Chemical Pathology), Department of Chemical Pathology, PO Box 227, Medunsa 0204, South Africa. Tel- 012-5214253, Fax- 012-5214157, Email- [email protected] KHINE ET AL.

introDuction

Nyaope, also known as whoonga, is a dangerous and highly addictve South Af- rican street drug (Department of Justce and Consituton SA, 2014). Nyaope is a fne brown powder due to its mixture with soil, sand or in some cases cement pow- der in order to disguise the underlying white power of drug of abuse. It is usually Figure 1. Nyaope sample wrapped in marijuana (dagga) leaves and smoked. It is not always clear what all of Nyaope frst appeared in the townships the ingredients of Nyaope are, and the in- around Durban in 2010, but is reportedly gredients may vary from sources of sellers. moving to impoverished areas around But one thing is clear: Nyaope is very ad- South Africa. Nyaope is a serious threat dictve and there are assumptons that it to South Africa’s HIV-positve populaton. may include heroin, detergent powder, rat There have been reports of gangs robbing poison, and crushed ant-retroviral drugs HIV/AIDS clinics in Soweto to obtain ARVs (ARVs) (Facts for Nyaope, 2014). ARVs are for making Nyaope, as well as addicted the medicatons used to treat patents users mugging ARV patents to obtain the with HIV. In most users, Nyaope not only drugs for themselves. It also promotes gives strong addicton but also severe ab- drug resistance to ARVs which has a huge dominal pain and seizures which are with- impact on the treatment programs for drawal symptoms. With respect to drug to patents living with HIV/AIDS (“Facts for drug interacton between various drugs of Nyaope,” 2014). Since Nyaope appears abuse, clinical studies showed that they similar to soil or cement powder, it is aggravate the body’s addictve reacton not easy to apprehend the users, sellers and also causes untoward efects such as or distributors. Analysis of Nyaope is not acute abdominal pain, seizures, vomitng, done routnely at medical laboratories or salivaton, psychosis etc (Bruce, Altce, forensic laboratories due to the high cost & Friedland, 2008). This makes the user and complexity of conventonal chroma- consume more and more Nyaope due to tography methods and lack of experience the belief that consuming more would re- in dealing with materials of such nature. lieve these symptoms. Nyaope is relatvely The newly introduced TOF-MS method cheap - about R20 to 30 for one “hit” and uses direct sample analysis and recent a person can become highly addicted af- studies showing its ability in identfying ter using the drug only once (Department individual compounds in the simulated of Justce and Consituton SA, 2014.). A mixture of drugs encourages the re- user will soon feel as if he needs several searchers to test its ability in the real drug hits to make it through the day. Untl and mixture samples (Wilhide, Lacourse, & unless the exact nature of the contents of Crowe, 2013) this street drug is understood, treatment The objectves of this study were to strategies and rehabilitaton support for identfy the exact consttuents of Nyaope the users may be difcult to achieve. by comparing the performance of two

50 COMPOSITION OF NYAOPE analytcal methods, namely the conven- Research and Development Laboratory tonal GC-MS (Gas Chromatography Mass in Midrand. All specimens were kept at Spectrometry) and the new TOF-MS (Time room temperature and cool dry place of Flight Mass Spectrometry). during transportaton to the laboratories.

sample preparaton before analysis MAteriAl AnD MethoDS Gas Chromatography coupled Mass Spectrometry (GC-MS) is an internaton- Ethical approval for this study was ob- ally accepted analytcal comparatve tained from the Medunsa Research Ethics technique where compounds are sepa- Commitee (MREC/H/165/2012:IR). The rated in the gas phase and a characteristc research method applied was a cross-sec- patern (mass spectrum) of the separate tonal descriptve and comparatve study. compounds is obtained. Sample to be analysed, in a liquid form is injected into Sample an inert gas stream (helium/argon) and Nyaope samples were randomly col- swept into a column packed with a sta- lected through the users from the town- tonary phase inside the oven. Absorptve ships and urban areas surrounding Preto- interacton between the components in ria in North Gauteng. The samples were the gas stream and the coatng leads to a collected over a period of one week from diferental separaton of the components 12 areas in Gauteng province (with the of the mixture, which are then swept to number of samples collected from each the detector which is a Mass Spectrom- area shown in brackets); Pretoria central etry. Mass Spectrometry takes injected (5), Sunnyside (4), Mamelodi (10), Bronk- material, ionizes it in a high vacuum, pro- horstspruit (3), Delmas (2), Winterveld pels and focuses these ions and their frag- (2), Ramogodu (3), Springs (1), Tembisa mentaton products through a magnetc (4), Garankuwa (2), Soshanguve (2) and mass analyser and then collects and mea- Witbank (2). However, obtaining Nyaope sures the amounts of each selected ion samples was a challenge as it requires in a detector. When the interested com- building a relatonship with the current pounds are not volatle in gas, the process users in order to build their trust and ob- of sample derivitzaton has to be done tain their assistance. which is tme consuming and the need to Samples were collected in the sterile purchase the reference material for each 30 ml specimen jars and were labelled compound in the mixture makes the test- with the name of the area and the num- ing very expensive (Foltz, Fentman, & ber. Each specimen was thoroughly mixed Foltz, 1980) before dividing it into two containers. The In the TOF-MS method, a PerkinElmer frst container was marked A and the sec- FlexarTMFX-15 LC pump with AxIONTM ond one B. The lids were screwed tghtly TOF MS was used for analysis. It uses to prevent any leakage or contaminaton. direct sample analysis with no need for All samples labelled A were analysed at sample preparaton except extracton the GC-MS laboratory at the SAPS foren- in liquid Methanol for the organic com- sic toxicology and the specimens labelled pounds. The supernatant was analysed B were analysed at the Perkin Elmer through Time of Flight instrument by di-

51 KHINE ET AL. rect applicaton on the specimen grid. The eas under the peaks may be used to est- extracted specimens are carried through mate the amount of compound present. the TOF path by the Nitrogen gas fow Table 1 represents the qualitatve detec- in a fxed temperature and gas pressure ton of the various compounds found in environment. The calibrator, Acetonitrile the Nyaope samples that were analysed. liquid was used for checking the resolu- Although multple samples were collect- ton of this compound in an expected po- ed from most areas, representaton of siton and gamma hydroxyl butyric acid only one sample was chosen based on the was used as a blank to check the baseline highest number of compounds present. of separaton and peak areas (Daugherty Samples of Tembisa and Winterveld & Crowe, 2014) were not sufcient to share for GC-MS Molecular identfcaton of each com- laboratory hence we could not report the pound in the mixture is achieved by its spectra of drugs for these two areas done mass/charge (m/z) rato which is matched by GC-MS although the analyses of these to the best ft m/z in the isotopic paterns samples were done on the TOF-MS. of known reference compounds pre- Figure 4 represents the mass spectra of stored in the instrument sofware library. both methods for the specimen collected The sensitvity of the analysis is M/Z 100- in Mamelodi Township as an example. In 1000 and error was < 1 ppm (parts per TOF-MS, calibrator’s positon is checked million). The direct sample applicaton and correlate with its isotopic patern part can be removed and replaced by and molecular mass. Then other organic HPLC (High Performance Liquid Chroma- compounds including drugs of abuse are tography) for separaton of compounds identfed in the same way. such as drug of abuse in more complex biological fuids (blood or urine) (Daugh- erty, 2011) DiScuSSion

GC-MS method could report the com- reSultS pounds quanttatvely if the reference materials or internal standards are used Both GC-MS and TOF-MS could report for calibraton of concentratons against the compounds qualitatvely although ar- m/z ratos of ionized molecules. However

Figure 2. From lef to right, direct sampling port, Time of Flight piece of the instrument TOF-MS and picture of sample applicaton on to the disposable wire mash.

52 COMPOSITION OF NYAOPE

Table 1. Consttuents of Nyaope samples acquired from various townships analysed by two diferent mass spectrometers

CONSTITUENTS IDENTIFIED IN NYAOPE

Heroine Heroine Method Method Cafeine Cafeine of sources of sources Citrofex A Citrofex Dimenoxitol Dimenoxitol Thiofentanyl Thiofentanyl Amphetamine / Amphetamine Acetaminophen Acetaminophen Phenobarbitone Phenobarbitone Benzodiazepines Benzodiazepines Opiate (meconin, (meconin, Opiate Area and number Area Dextromethophan Dextromethophan meth- metabolites metabolites meth- Moramide narcotcs narcotcs Moramide Duracaine/lidocaine Duracaine/lidocaine Codeine/metabolites Codeine/metabolites Cathine (b OH amphet) (b OH amphet) Cathine Fenethyline (stmulant) (stmulant) Fenethyline Morphine /metabolites Morphine /metabolites .papaverine, methadone.papaverine, Benzitramide (narcotcs) (narcotcs) Benzitramide Pipradol (Dopamine reupt) (Dopamine reupt) Pipradol Ant-retroviral (zidovudine) (zidovudine) Ant-retroviral

Garankuwa GC + + + + + + + (sample 2) TOF + + + + + + +

Soshanguve GC + + + + + (sample1) TOF + + + + +

Bronkhorstspruit GC + + + + + (sample 2) TOF + + + + + + + + + +

Witbank GC + + + + + + + (sample 2) TOF + + + + +

Mamelodi GC + + + + + (sample 8) TOF + + + + + + + + + +

Springs GC + + + + + + (sample 1) TOF + + + + + +

Pretoria central GC + + + + + + + (sample 5) TOF + + + + + + + +

Ramogodu GC + + (sample 3) TOF + + + + + + +

Winterveld GC Not done due to insufcient samples (sample 2) TOF + + + + + + +

Delmas GC + + + + + (sample 2) TOF + + + + + + +

Tembisa GC Not done due to insufcient samples (sample 4) TOF + + + + + + + + +

Sunnyside GC + + + + + (sample 9) TOF + + + + + + + + +

53 KHINE ET AL.

TOF-MS: average spectrum

GC-MS:

Figure 3. Example of Mass Spectra for one selected Nyaope specimen collected in Mamelodi township of Pretoria using isotopic internal standards are rela- phetamine (MDA) and Heroine. A local tvely more stable than the natural com- anaesthetc Duracaine/lidocaine was also pound but they are more costly (Karacic found in some samples which It has a & Skender, 2000). Another study showed mood enhancing efect. Detected by the analysis of drugs of abuse in hair by LC- TOF-MS but not by GC-MS were the ant- MS method (liquid chromatography) and biotc (citrofex) and the ARV zidovudine, it also uses expansive reference com- CNS depressants such as phenobarbitone pounds, accessories and the method is and benzodiazepines, benzitramide, mo- complex requiring highly skilled opera- ramide intermediates and thiofentanyl, tors (Paterson, Mclachlan-troup, Cordero, stmulants such as Pipradol, and feneth- Dohnal, & Carman, 2001). In our study, yline. This could be due to the fact that to compare the performance of the two GC-MS system requires reference materi- methods, we reported results qualitatve- als of each of these compounds to verify ly from both GC-MS and TOF-MS systems. and discriminate them from other com- Consistently seen in all samples were pounds. Of note, , the cafeine, drugs of abuse such as opiates, anttussive cough suppressant was de- codeine, morphine, methyl-dioxy am- tected by both systems.

54 COMPOSITION OF NYAOPE

Resoluton of unidentfable peaks is due to its higher specifcity to detect a challenging for both methods. In TOF-MS, partcular suspected compound/drug in a the unknown peak can be matched in the sample and also ability to report in quan- instrument sofware library or on internet ttatve level provides a platorm for con- based on correlaton of molecular mass frmaton of presence of a drug of abuse and isotonic patern and aim for more which is required for medico-legal pro- than 95% of match. In order to facilitate cedures. Comparatvely, the consumable the accuracy of identfcaton, drugs of cost for TOF-MS is R 10 for a disposable abuse artfcially made up in a mixture sample grid. Each specimen uses 500 mi- (without any soil, sand or cement like in cro litre of Methanol for extracton before Nyaope) was analyzed on TOF-MS by Per- analysis. With this relatvely inexpensive kin Elmer to identfy and save the isotopic running cost and faster analytcal tme, paterns in the instrument library (Wil- TOF- DSA MS provides an opportunity for hide et al., 2013) (Robinson, 2010). This screening of drugs and compounds in a makes the system possible to match 100% mixture of unknown whereas the GC-MS ftness with the paterns received from system is more useful in confrmaton of a analysis of Nyaope samples. Confrmaton partcular screened positve compound in of identfed peaks is much efcient with the same mixture. GC-MS system as it can fragment the peak Contrary to prior assumptons, our into various ions which show a specifc samples did not show any inclusion of rat patern of mass and charge rato however poison but the ant-retroviral drug was a reference material is needed to discrim- found to be present in few samples of inate this patern from the closely related two areas. This has shed the light on per- other compounds for confrmaton(Zhao ceptons and beliefs of users who are also & Zhang, 2012) living with HIV/AIDS in these areas and Regarding the analytcal runtme, the townships in their adherence problems TOF-MS produces results in 15 seconds due to poor tolerance to the side efects for a mixture as opposed to GC-MS which of ARV therapy. Consumpton of ARVs in takes 10 minutes for each compound. such inhalatonal form may enhance drug Time form collecton to reportng of re- resistance or damage to the respiratory sults marks the turnaround tme and it mucosa although no study has been done may depend on the workload and speci- to prove this. This important fnding has men batching requirement of the testng an impact on the HIV treatment programs laboratory for cost-efectveness on the natonwide and has to be taken into con- reagents and reference materials espe- sideraton when counselling for adher- cially for the GC-MS lab. ence is provided for patents living with Cost-wise, GC-MS is more expensive HIV. due to high cost of reference material and the need to perform specimen derivitsa- ton for certain compounds. It costs ap- concluSion proximately R 560 for quanttatve drug assay per sample according to the scale of Identfying the consttuents of Nyaope beneft guideline for South Africa. How- is important in order to select appropri- ever, the discriminatory power of GC-MS ate antdotes for the treatment of with-

55 KHINE ET AL. drawal symptoms during rehabilitaton as News Leter, Retrieved Aug 24, 2014 well as providing evidence for criminaliza- from htp://www.justce.gov.za ton of dealers and users. The challenge Facts for Nyaope. (2014). HIV South Af- is the heterogeneity and constant change rica, mobilizaton. Retrieved Aug 25, in its formula depending on the availabil- 2014 from htp://hivsa.mobi/h4l/art. ity of consttuents in the community. For php?art=147 unknown drug mixtures such as Nyaope, Foltz, R. L., Fentman, A. F., & Foltz, R. B. TOF- DSA MS provides a beter platorm (1980). GC / MS assays for abused for the inital screening of consttuents drugs in Body Fluids. Monograph, Na- while GC-MS provides a confrmatory tonal Insttute for Drug Abuse, USA. testng of a specifc compound indicated Karacic, V., & Skender, L. (2000). Analy- as screen positve in the TOF-MS. sis of Drugs of Abuse in urine by Gas Chromatography/Mass Spectrom- etry: Experience and Applicaton. aCkNOwlEDgEMENTs Archive in Radiaton and Toxicology, 2000;51:389–400, 51, 389–400. We thank SAPS Toxicology laboratory Paterson, S., Mclachlan-troup, N., Cor- and Perkin Elmer SA Laboratory for analy- dero, R., Dohnal, M., & Carman, S. sis of Nyaope samples. (2001). Qualitatve Screening for Drugs of Abuse in Hair Using GC- referenceS MS. Journal of Analytcal Toxicology, 25(April), 203–208. Bruce, R. D., Altce, F. L., & Friedland, G. H. Robinson, K. (2010). AxION DSA: Qual- (2008). Pharmacokinetc drug interac- ity data without chromatography or tons between drugs of abuse and an- sample preparaton. Perkin Elmer Re- tretroviral medicatons: implicatons port and management for clinical practce. Wilhide, J., Lacourse, W. R., & Crowe, Expert Review in Clinical Pharmacol- H. (2013). Confrmaton of drugs of ogy, 1(1), 115–128. abuse in pseudo drug mixes using Daugherty, S. (2011). Direct sample anal- HPLC- ESI TOF MS. Poster, American ysis using Time of Flight Mass Spec- Associaton of Clinical Chemistry an- trometry. PerkinElmer Report. nual congress. Daugherty, S., & Crowe, H. (2014). Auto- Zhao, S., & Zhang, R. (2012). A Rapid Meth- mated Direct Sample Analysis (DSA od for Detecton of Drugs of Abuse / TOF) for the Rapid Testng of Drug in Blood Samples Using the Thermal Compounds. Perkin Elmer Report Separaton Probe and the 5975T LTM Department of Justce and Consituton, GC / MS. Applicaton Note.Insttute of S. A. (2014). THE END FOR NYAOPE! Forensic Science, Shanghai, China.

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