New psychoactive substances (NPS) reach the recreational drugs market at a fast pace and are of concern because of potential health risks. In addition to not being legally regulated, NPS escape detection in standard drug tests. Drug testing laboratories, therefore, must adapt their analytical methods to also cover these new substances. For screening and confirmation of NPS, mass-spectrometric multicomponent methods are useful.
by Prof. Olof Beck and Prof. Anders Helander
New psychoactive substances
The emergence of new drugs of abuse that are designed to circumvent narcotics legislation by slight chemical structural modifications of already classified drugs represents an ever increasing problem [1, 2]. Nowadays, this phenomenon is commonly termed ‘new psychoactive substances’ or ‘NPS’, but also other names such as designer drugs, legal highs, research chemicals, smart drugs, bath salts, and spice have been and are used. The NPS problem is of global concern but may vary in extent between countries, partly due to national differences in legislation and drug culture. Statistics from the EU Early Warning System operated by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and Europol on the number of NPS reported for the first time in Europe on a yearly basis gives a good insight on the progress of this phenomenon (Fig. 1) [2]. Over the past 6 years particularly, it has escalated to the level of more than 100 new substances in 2014 (i.e. about two new substances each week on average). The NPS market was long dominated by stimulants and synthetic cannabinoids but currently comprises all classes of abused substances [2].
Problems related to NPS
NPS are of particular concern because they can be sold openly in web-based shops and elsewhere and thereby reach new drug users that are attracted by their ‘legal’ status. Of public concern are the unforeseen toxic effects of NPS, as using these uncontrolled and unsafe substances and products may lead to severe intoxication and even death [1, 3]. In Sweden, the progress of the NPS phenomenon and associated harmful effects has been followed in a collaborative project between the Department of Laboratory Medicine at the Karolinska University Hospital and the Karolinska Institutet, and the Swedish Poisons Information Center [3, 4]. This project, named STRIDA, enrolls patients with suspected NPS intoxication presenting in emergency departments all over the country. By combining the results from laboratory investigations of serum and urine samples with clinical information, new knowledge about NPS prevalence and toxicity is compiled. Since the start in 2010, the STRIDA project has documented over 2000 non-fatal but often severe acute intoxication cases involving a large number of different NPS. Polydrug use is commonly seen in these cases [3].
NPS in drug screening
One reason for using NPS instead of conventional drugs of abuse may be that NPS often remain undetected in standard drug testing procedures. Accordingly they are especially attractive alternatives for individuals who want to minimize the risk of being detected, such as in workplace drug testing and drug rehabilitation programmes.
The established procedure for drug testing is to use initial screening by immunoassays and then to confirm positive samples using methods based on the more sensitive and selective mass spectrometry (MS) technique. On one hand, the NPS present a challenge for the immunoassay screening, as available methods are typically directed only towards the conventional substances, e.g. amphetamines (amphetamine and methamphetamine), tetrahydrocannabinolcarboxylic acid (THC, cannabis), morphine (heroin), and benzoyl ecgonine (cocaine). On the other hand, as NPS are often designed to mimic and are chemical derivatives of conventional drugs, there is a possibility that certain NPS will also bind to (i.e. cross-react with) the antibodies used in immunoassay screening methods. And this is indeed the case. However, when these ‘false-positive’ screening results are subjected to confirmatory analysis by methods based on MS detection, they will turn out negative (i.e. ‘false-negative’ for drug use), if the MS method is only directed toward the standard set of abused drugs.
Cross-reactivity of NPS in immunoassays
When ecstasy (3,4-methylenedioxymethamphetamine, MDMA) became established as a street drug, interest emerged to detect it in immunoassay screening. MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) were found to be detectable in existing assays for amphetamine and methamphetamine, due to a high degree of cross-reactivity for these compounds [5]. Likewise, also other new amphetamine-like substances were detectable [6].
However, although many NPS showed low cross-reactivity in commercial immunoassays [7, 8], the stimulant methylenedioxypyrovalerone (MDPV) was reported to cross-react in the CEDIA phencyclidine test [9]. A study from the authors’ laboratory comprising 45 NPS confirmed that several possessed chemical similarities leading to high cross-reactivity in the immunochemical screening tests commonly employed in routine urine drug testing [10]. The detectability of NPS observed to possess cross-reactivity was further confirmed by analysis of urine specimens from authentic intoxication cases included in the STRIDA project (Table 1). Given a more widespread use of new drugs among individuals subjected to drug testing, an increased number of unconfirmed positive screening results may occur.
The cross-reactivity for NPS in current screening assays may be seen as a problem or as a possibility to detect more substances. One possibility for improved drug testing is to include the most common new substances in the confirmation methods. As ecstasy became established as an illicit drug, new immunochemical screening tests for amphetamine/methamphetamine were developed that also included MDMA and MDA. Authentic case samples were used to demonstrate the capability of several commercial amphetamine class screening tests to detect MDMA/MDA. At that time, cross-reactivity towards the new ‘amphetamine’ analytes was wanted [5]. With the advent of the large number of NPS, both legal and illegal, the strategy to also cover new substances in the screening assays for classical narcotic drug substances may not be feasible. For example, the multitude of new synthetic cannabinoids (‘spice’) have not been incorporated in screening tests for THC, but resulted in the development of new independent tests [11].
One approach put forward to understand the potential of immunoassays to detect NPS is to use molecular similarity models [12]. Interestingly, the work of Petrie and co-workers [13] included such a molecular modelling method to predict the cross-reactivity of 261 amphetamine-like compounds. However, when comparing the theoretical data with our experimental data for one compound, the predicted reactivity for butylone was 10 times lower than that observed. In a more recent publication, it was proposed that molecular similarity models could be used to design new immunoassays with sensitivity for a larger number of target compounds [14].
NPS analysis by mass spectrometry
Another analytical strategy to cover NPS in drug testing is to employ MS-based ‘screening’ methods. As part of the STRIDA project, a multicomponent analytical MS method for NPS analysis in urine and serum specimens has been developed [15]. The method uses MS in combination with liquid chromatography (LC-MS/MS in selected-reaction monitoring mode) and is continuously updated as new NPS appear. There are also other methods for multicomponent screening of drugs in urine and plasma/serum, which proves that this technology can be employed in routine drug testing [16].
The LC-MS/MS technique has great potential for drug testing and for clinical laboratories in general. There are examples of laboratories that have already successfully replaced immunoassay screening by MS methods, also for the conventional drugs of abuse [17]. One way to make this possible and cost-effective is to use simple sample preparation procedures, e.g. a simple dilution of urine with internal standards [16]. When studying the cross-reactivity of 30 NPS in commercial ELISA tests for serum and blood, only a few were found to display cross-reactivity, and it was therefore proposed that MS methods should be used in future drug screening [18]. One attraction of MS-based screening is that accurate results are already obtained from the initial analytical step, which may be especially important in cases of acute intoxication (Fig. 2).
Potential of high-resolution MS
One promising technique for drug screening is high-resolution MS (HRMS) [19]. In the HRMS technique, the acquisition of data can be made with an untargeted design. Thousands of substances can be monitored at the same time without the need for optimizing MS parameters for each compound. In addition, new compounds can be searched for retrospectively.
Conclusion
The NPS present a challenge for drug testing laboratories and calls for novel drug screening strategies. It is likely that the current broader spectrum of abused psychoactive drugs will persist in at least in the foreseeable future. This new drug situation has put the performance of drug testing into focus and indicates that drug testing laboratories will play a more important role, as on-site drug screening using dipsticks is likely to lose significance.
References
1. Lewin AH, Seltzman HH, Carroll FI, Mascarella SW, Reddy PA. Emergence and properties of spice and bath salts: A medicinal chemistry perspective. Life Sci. 2014; 97: 9–19.
2. EMCDDA. New psychoactive substances in Europe. An update from the EU Early Warning System (March 2015). 2015. Available at: http://www.emcdda.europa.eu/attachements.cfm/att_235958_EN_TD0415135ENN.pdf.
3. Helander A, Bäckberg M, Hultén P, Al-Saffar Y, Beck O. Detection of new psychoactive substance use among emergency room patients: results from the Swedish STRIDA project. Forensic Sci Int. 2014; 243: 23–29.
4. Helander A, Bäckberg M, Beck O. MT-45, a new psychoactive substance associated with hearing loss and unconsciousness. Clin Toxicol. 2014; 52(8): 901–904.
5. Hsu J, Liu C, Hsu CP, Tsay WI, Li JH, Lin DL, Liu RH. Performance characteristics of selected immunoassays for preliminary test of 3,4-methylenedioxymethamphetamine, methamphetamine, and related drugs in urine specimens. J Anal Toxicol. 2003; 27: 471–478.
6. Apollonio LG, Whittall IR, Pianca DJ, Kyd JM, Haher WA. Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using Bio-Quant direct Elisa presumptive assays for amphetamine and methamphetamine. J Anal Toxicol. 2007; 31: 208–213.
7. Kerrigan S, Mellon MB, Banuelos S, Arndt C. Evaluation of commercial enzyme-linked immuno assays to identify psychedelic phenethylamines. J Anal Toxicol. 2011; 35: 444–451.
8. Bell C, George C, Kicman AT, Traynor A. Development of a rapid LC-MS/MS method for direct urinalysis of designer drugs. Drug Test Anal. 2011; 3: 496–504.
9. Macher AM, Penders TM. False-positive phencyclidine immunoassay results caused by 3,4-methylenedioxypyrovalerone (MDPV). Drug Test Anal. 2012; 5: 130–132.
10. Beck O, Rausberg L, Al-Saffar Y, Villen T, Karlsson L, Hansson T, Helander A. Detectability of new psychoactive substances, ‘legal highs’, in CEDIA, EMIT, and KIMS immunochemical screening assays for drugs of abuse. Drug Test Anal. 2014; 6: 492–499.
11. Arntson A, Ofsa B, Lancaster D, Simon JR, McMullin M, Logan B. Validation of a novel immunoassay for the detection of synthetic cannabinoids and metabolites in urine specimens. J Anal Toxicol. 2013; 37: 284–290.
12. Krasowski MD, Pizon AF, Siam MG, Giannoutsos S, Iyer M, Ekins S. Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine. BMC Emerg Med. 2009; 9: 5.
13. Petrie M, Lynch KL, Ekins S, Chang JS, Goetz RJ, Wu AHB, Krasowski MD. Cross-reactivity studies and predictive modeling of “Bath Salts” and other amphetamine-type stimulants with amphetamine screening immunoassays. Clin Toxicol. 2013; 51: 83–91.
14. Krasowski MD, Ekins S. Using cheminformatics to predict cross reactivity of “designer drugs” to their currently available immunoassays. J. Cheminform. 2014; 6: 22.
15. Al-Saffar Y, Stephanson NN, Beck O. Multicomponent LC-MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine – experience from the Swedish population. J Chromatogr B 2013; 930: 112–120.
16. Beck O, Ericsson M. Methods for urine drug testing using one-step dilution and direct injection in combination with LC-MS/MS and LC-HRMS. Bioanalysis 2014; 6 : 2229–2244.
17. Eichhorst JC, Etter ML, Rousseaux N, Lehotay DC. Drugs of abuse testing by tandem mass spectrometry: A rapid, simple method to replace immunoassays. Clin Biochem. 2009; 42: 1531–1542.
18. Swortwood MJ, Hearn WL, DeCaprio AP. Cross-reactivity of designer drugs, including cathinone derivatives, in commercial enzyme-linked immunosorbent assays. 2014; 6: 716–727.
19. Maurer HH. What is the future of (ultra) high performance liquid chromatography coupled to low and high resolution mass spectrometry for toxicological drug screening? J Chromatogr A 2013; 1292: 19–24.
The authors
Olof Beck*1,3 PhD and Anders Helander2,3 PhD
1Department of Clinical Pharmacology, Karolinska University Laboratory Huddinge, Sweden
2Department of Clinical Chemistry, Karolinska University Laboratory Huddinge, Sweden
3Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
*Corresponding author
E-mail: olof.beck@karolinska.se
Many people become infected with hepatitis C virus (HCV) every year and these infections often have no symptoms. A significant number of patients will go on to develop chronic liver disease and potentially hepatocellular carcinoma. Early detection of HCV infection is of great importance, but remains challenging. This article describes the advantages and limitations of methods of HCV diagnosis.
by Dr O. Blach, Dr D. Lawrence, Dr F. Cresswell and Prof. M. Fisher
Hepatitis C virus infection
It is estimated that 3% of the world’s population has hepatitis C virus (HCV), with a further 3–4 million people becoming newly infected every year [1]. Early detection of HCV infection is of great importance, as prompt diagnosis enables contact tracing, partner notification, health promotion advice to reduce the risk of onward transmission and disease progression, and the opportunity for early treatment, which may offer the best opportunity for cure [2].
However, diagnosing acute hepatitis C remains challenging. Most patients with acute infections are asymptomatic, and even when symptoms are present, they are often non-specific, not severe, and may not present in the same way as those with other hepatitis viruses (such as A, B and E). Approximately 10–20% of patients clear the virus spontaneously during acute infection; the remainder progress to chronic infection which, if unrecognized, will progress in a significant proportion to chronic liver disease, cirrhosis, end-stage liver disease and hepatocellular carcinoma [2]. Although newer directly acting antiviral drugs (DAAs) against HCV will transform management, for many individuals pegylated interferon and ribavirin may remain standard of care for some time until these can be afforded. Therefore early diagnosis for many will offer the best opportunity for cure at the present time.
Established diagnostic tests
The diagnosis of acute hepatitis C is usually made after detection of abnormal liver function tests or on routine screening in specific populations, such as those with HIV infection, on hemodialysis for end-stage renal failure, or accessing services for injecting drug users. Traditionally, seroconversion from anti-HCV antibody (anti-HCV) negative to positive, a process which takes places around 12 weeks after infection, is detected by enzyme-linked immunosorbent assays (ELISA, EIA) or chemiluminescence immunoassay (CIA) [3].
However, although the presence of anti-HCV indicates infection with HCV at some point, it does not determine whether it is acute, chronic or resolved. Furthermore, anti-HCV may not be detectable during this aforementioned 12-week ‘window period’, or if the patient is immunocompromised and therefore has an impaired ability to produce antibodies [4], with delayed seroconversion up to 18 months being reported [5].
Detection of viremia in the setting of a negative anti-HCV (during the seronegative ‘window period’), and therefore verification of active HCV infection has historically been done using nucleic acid amplification test (NAAT) for HCV RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR), which can detect HCV RNA in serum 1–3 weeks after infection [6–8]. Although the ‘gold standard’ for diagnosing acute HCV infection, HCV qRT-PCR has several shortcomings: it is costly, labour-intensive, time-consuming and requires advanced technical skills, separate facilities (separate platform) and equipment [9], which make it particularly impractical in a resource-poor setting. As a consequence, HCV core antigen (Ag) quantification as a surrogate marker of HCV replication has been suggested as an alternative assay for initial testing of acute hepatitis [10].
HCV core antigen
HCV core Ag is part of the HCV capsid formed by core protein polymerization, and as such, is one of the best ‘conserved’ products of viral genome [11]. Using HCV core Ag, acute infection with HCV can be detected in the serum earlier than with the current anti-HCV screening assays [12], and only 1–2 days later than with HCV RNA NAAT tests [13].
Since the development of the first HCV core Ag tests around 2000, newer assays, which are up to 25 times more sensitive, have become available and are licensed in several countries. A recent meta-analysis of 25 studies conducted by Gu et al. [14] compared the diagnostic accuracy of HCV core Ag (index reference) with HCV RNA (‘gold standard’) and showed good pooled sensitivity of 0.84 (95% CI, 0.83–0.85), with excellent pooled specificity of 0.98 (95% CI, 0.97–0.98) for HCV core Ag assays. HCV core Ag can therefore be used as a marker of viraemia [7] with the lower limit of detection corresponding to HCV RNA load of 700–1100 IU/mL [15]. Positive and negative predictive values reported in the literature for HCV core Ag assays are also high, with one study reporting PPV of 100% and NPV of 97% [16]. However, re-testing samples with low positive Ag values (<35fM) has been recommended after one study by Shepherd et al. [17] reported 37% false positive rates with such results. Another study by Cresswell et al. [7] recorded two false-indeterminate results, one of which was false positive on re-testing.
Furthermore, HCV core Ag levels closely track those of HCV RNA with multiple studies identifying a strong non-linear correlation between the two, thus potentially also allowing clinical monitoring of a patient’s therapy, independently of HCV genotype [15]. This is mainly the case in samples with HCV RNA levels above 20 000 IU/ml, thereby limiting their use in practice [18].
Given its slightly lower sensitivity compared with HCV RNA PCR, the utility of HCV core Ag testing in a diagnostic algorithm for acute hepatitis C is dependent on the practicalities of testing in a given population setting and the potential cost savings [19]. One appealing advantage of HCV core Ag assays lies in the potential for reflex HCV core Ag testing in anti-HCV positive samples using the same testing platform and the same sample [20], thus providing physicians with clinically meaningful results of both anti-HCV and HCV core Ag within an hour.
HCV core Ag could also prove to be more stable than HCV RNA in situations where testing cannot be done on a fresh sample or where a sample needs to be transported to another laboratory [21]. As such, HCV antigen detection could be a viable next step following a positive anti-HCV test, and additional HCV RNA testing would only be necessary with negative or low positive HCV core Ag values.
Furthermore, besides a faster processing time compared to traditional molecular tests, HCV core Ag assays are cheaper [22] and thereby especially attractive in low-resource settings or where PCR may be unavailable [9]. Cresswell et al. estimated potential cost savings of $18 275 in equipment and $6964 in manpower per year in an HIV cohort of 2200 people, had HCV core Ag been used in place of HCV RNA PCR [7].
Special populations
The usefulness of HCV core Ag as a screening investigation in the immunocompromised cohort has attracted considerable attention recently, given their impaired antibody production and the well-recognized delay in HCV antibody seroconversion [5]. High sensitivities (100%) and specificities (97.9 and 97.7%) were reported by Cresswell et al. [7] and Carney et al. [23] in diagnosing acute hepatitis C in HIV-infected individuals by HCV core Ag. Another study of dialysis patients by Moini et al. found only one HCV RNA positive patient to be HCV core Ag negative (note a low HCV viral load of <100 IU/mL) [24].
Finally, in the context of blood transfusion or organ transplantation, the modern HCV RNA assays remain the most sensitive and preferred option [25], but in a resource-limited blood bank setting, testing with HCV core Ag might be superior to no testing for HCV viremia at all. Further research is needed to determine the role of HCV core Ag testing in monitoring of both the untreated patients and those undergoing therapy, as well as in predicting the histological chances and disease progression.
Looking to the future
In conclusion, given the inadequacies of HCV antibody testing in acute infection and the time and financial constraints of HCV RNA PCR, HCV core Ag detection offers a new, cheaper and effective way of testing for acute hepatitis C, and is a promising confirmatory test for anti-HCV positive patients. Given the emerging evidence on the constantly improving HCV core Ag assays, we believe that national guidelines should now begin to consider HCV core Ag testing as an integral part of the HCV screening algorithm for acute HCV infection, as illustrated in Figure 1.
References
1. World Health Organization. Secretariat. Viral hepatitis. Sixty-Third World Health Assembly A63/15. Provisional agenda item 11.12. 25 March 2010. World Health Organization, 2010.
2. Webster DP, Klenerman P, Collier J, Jeffery KJ. Development of novel treatments for hepatitis C. Lancet Infect Dis. 2009; 9(2): 108–117.
3. Pondé RA. Enzyme-linked immunosorbent/chemiluminescence assays, recombinant immunoblot assays and nucleic acid tests in the diagnosis of HCV infection. Eur J Clin Microbiol Infect Dis. 2013; 32(8): 985–988.
4. Chamot E, Hirschel B, Wintsch J, et al. Loss of antibodies against hepatitis C virus in HIV-seropositive intravenous drug users. AIDS 1990; 4(12): 1275–1277.
5. Thomson EC, Nastouli E, Main J, et al. Delayed anti-HCV antibody response in HIV-positive men acutely infected with HCV. AIDS 2009; 23: 89–93.
6. Cox AL, Netski DM, Mosbruger T, et al. Prospective evaluation of community-acquired acute-phase hepatitis C virus infection. Clin Inf Dis. 2005; 40: 951–958.
7. Cresswell F, Fisher M, Hughes D, et al. Hepatitis C core antigen testing: a reliable, quick, and potentially cost-effective alternative to hepatitis c polymerase chain reaction in diagnosing acute hepatitis C virus infection. Clin Inf Dis. 2015; 60(2): 263–266.
8. Umar M, Khan A, Abbas Z, et al. World Gastroenterology Organisation global guidelines: diagnosis, management and prevention of hepatitis C April 2013. J Clin Gastroenterol. 2014; 48(3): 204–217.
9. Chakravarti A, Chauhan MS, Dogra G, et al. Hepatitis C virus core antigen assay: can we think beyond convention in resource limited settings? Braz J Infect Dis. 2013; 17(3): 369–374.
10. Hadziyannis E, Minopetrou M, Georgiou A, et al. Is HCV core antigen a reliable marker of viral load? An evaluation of HCV core antigen automated immunoassay. Ann Gastroenterol. 2013; 26(2): 146–149.
11. Caruntu F, Benea L. Acute Hepatitis C Virus Infection: Diagnosis, Pathogenesis, Treatment. J Gastrointestin Liver Dis. 2006; 15(3): 249–256.
12. Dawson G. The potential role of HCV core antigen testing in diagnosing HCV infection. Antivir Ther. 2012; 17: 1431–1435.
13. Heathcote J, et al. World Gastroenterology Organisation Practice Guidelines: Management of acute viral hepatitis. (December 2003). http://www.worldgastroenterology.org/assets/downloads/en/pdf/guidelines/02_acute_hepatitis.pdf visited on 22nd Jan 2015.
14. Gu S, Liu J, Zhang H, et al. Core antigen tests for hepatitis C virus: a meta-analysis. Mol Biol Rep. 2012; 39: 8197–8208.
15. Medici MC, Furlini G, Rodella A, et al. Hepatitis C virus core antigen: analytical performances, correlation with viraemia and potential applications of a quantitative, automated immunoassay. J Clin Virol. 2011; 51: 264–269.
16. Li Cavoli G, Zagarrigo C, Schillaci O, et al. Hepatitis C Virus core antigen test in monitoring of dialysis patients. Hepat Res Treat. 2012; 2012: 832021.
17. Shepherd S, Aitken C, Walkowicz M, et al. HCV antigen testing in a busy diagnostic laboratory. Clinical Microbiology and Infection 2012; 18: 676–77.
18. Pawlotski JM. Use and interpretation of virological tests for hepatitis C. Hepatology 2002; 36(5,S1): S65–S73.
19. Tillmann H. Hepatitis C virus core antigen testing: role in diagnosis, disease monitoring and treatment. World J Gastroenterol. 2014; 20(22): 6701–6706.
20. Ottiger C, Gygli N, Huber AR. Detection limit of architect hepatitis C core antigen assay in correlation with HCV RNA, and renewed confirmation algorithm for reactive anti-HCV samples. J Clin Virol. 2013; 58: 535–540.
21. Miedouge M, Saune K, Kamar N, et al. Analytical evaluation of HCV core antigen and interest for HCV screening in haemodialysis patients. J Clin Virol. 2010; 48: 18–21.
22. Tedder RS, Tuke P, Wallis N, et al. Therapy-induced clearance of HCV core antigen from plasma predicts an end of treatment viral response. J Viral Hepat. 2013; 20: 65–71.
23. Carney R, Maranao D, Sudra R, et al. A hepatitis C virus core antigen assay is a cost-effective, sensitive and specific test in the detection of acute hepatitis C in HIV infected subjects. HIV Med. 2014; 15(S3): 8.
24. Moini M, Ziyaeyan M, Aghaei S, et al. Hepatitis C virus (HCV) infection rate among seronegative hemodialysis patients screened by two methods; HCV core antigen and polymerase chain reaction. Hepat Mon. 2013; 13: e9147.
25. Waldenström J, Konar J, Ekermo B, et al. Neonatal transfusion-transmitted hepatitis C virus infection following a pre-seroconversion window-phase donation in Sweden. Scand J Infect Dis. 2013; 45: 796–99.
The authors
Ola Blach*1 MBChB, BSc; David Lawrence1 MBChB, MSc; Fiona Cresswell1 MD, MBBS; and Martin Fisher1,2 FRCP, MBBS, BSc
1Lawson Unit, Department of HIV and Sexual Health, Royal Sussex County Hospital, Brighton, UK
2Brighton and Sussex Medical School, Brighton, UK
*Corresponding author
E-mail: ola.blach@doctors.org.uk
A simple and rapid method for simultaneous determination of mycophenolic acid (MPA) and its glucuronide (MPAG) in plasma using high-performance liquid chromatography (HPLC) with UV detection is described. MPA is an immunosuppressant used in kidney, liver and heart transplantation to prevent organ rejection. Owing to MPA’s narrow therapeutic window and considerable variability within and between patients, the routine monitoring of MPA concentrations is suggested.
by C. Misch and Prof. P. Tang PhD
Background
Mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS) are widely used to prevent organ rejection after organ transplantation. Following administration, both prodrugs are rapidly hydrolysed to mycophenolic acid (MPA), the active immunosuppressant. MPA is able to suppress the synthesis of guanosine nucleotides in T and B lymphocytes, principally via noncompetitive, selective and reversible inhibition of inosine monophosphate dehydrogenase. MPA is primarily metabolized by the uridine diphosphate glucuronyl transferase to an inactive glucuronide (MPAG), which is transported from liver into bile. Biliary MPAG then enters the gastrointestinal (GI) tract, where it is converted back to MPA, which is then recycled into the bloodstream via the enterohepatic circulation pathway. Several studies have documented that variation in MPA plasma concentrations are unpredictable and variability in plasma concentrations of MPA within and between individuals are high [1–4]. The highly variable set of patient situations on MPA therapies can cause variable risk for adverse effects such as hematologic and GI toxicity. Therapeutic drug monitoring (TDM) of MPA and MPAG can aid clinicians develop personalized therapy strategies to avoid toxicity and maintain efficacy.
For measuring MPA and MPAG concentrations in biological samples, high-performance liquid chromatography (HPLC) has been the primary technique. Scrutinizing all reported technologies, mass spectrometry is generally superior in sensitivity, selectivity and specificity to other detectors. However, the purchase, maintenance and running costs of mass spectrometry are high. From an economic standpoint, HPLC/UV methods [5–10] allow cost-effective assay while provide adequate sensitivity, selectivity and specificity for measuring clinically relevant concentrations of MPA (0.5–5 μg/mL) and MPAG (5–100 µg/mL). The intent of this application was to develop a simple and rapid HPLC/UV method for the determination of MPA and MPAG concentrations in plasma.
Experimental details
Apparatus and materials
The instrument and analytical conditions are listed in Table 1. MPA and internal standard clonazepam were obtained from Sigma (St. Louis, MO). MPAG was from TRC (Toronto Research Chemicals). All other chemicals used were analytical grade or HPLC grade. Separate stock solutions of clonazepam, MPA and MPAG were prepared by accurately weighing and dissolving it in an appropriate amount of methanol.
Calibration/sample preparation
For constructing calibration curves, the concentration ranges of MPA and MPAG were set to 0.1–20 and 1–200 µg/mL, respectively. To 0.1 mL of blank plasma, 0.1 mL each of clonazepam, MPA, MPAG and methanol were added; the mixture was vortex-mixed for 1 min. After centrifugation for 10 min at 10 000 rpm, the supernatant was transferred to an autosampler vial. To 0.1 mL of patient plasma, 0.1 mL clonazepam and 0.3 mL methanol were added and processed as stated above
Results and discussion
Chromatographic separation
A typical chromatogram is presented in Figure 1. These compounds resolved without any overlapping of their peaks or ambiguity in identification. All compounds were eluted within 14 min. No interference was observed in patient samples containing endogenous matrix components, metabolites, xenobiotics and concomitant medication (see Table 2).
Linearity
Good linearities (1/x weighted) were obtained for MPA and MPAG with coefficient of determination (r2) values >0.990 from 0.1 to 20 µg/mL (for MPA) or 1 to 200 µg/mL (for MPAG). The percentage deviation was <15%.
Method validation
Method accuracy and precision data are presented in Table 3. Overall the percentage recovery of MPA and MPAG ranged from 93 to 105%, indicating the consistent, precise, and reproducible extraction efficiency of the method. Both within-run (n=6) and between-run (n=30) precisions were <9%.
Comparison between two HPLC-UV methods
Figure 2a and 2b illustrate comparisons between the current method and reference method. The reference method was also based on a HPLC-UV procedure. The correlation between the two methods was good; the linear regression statistics indicated both r2 values >0.990 (P<0.0001). The linear regression equation for MPA correlation was y = 1.018 x + 0.031 with a standard error value of 0.24; where y, the current method and x, the reference method. The linear regression equation for MPAG correlation was y = 0.984 x − 0.292 with a standard error value of 5.08.
MPA and MPAG concentrations in plasma
Figure 3 illustrates considerable variability of MPA and MPAG concentrations in patient plasma. MPA concentrations ranged from 0.3 to143 µg/mL; MPAG concentrations ranged from 1.2 to 457 µg/mL; MPAG : MPA mole ratio ranged from 0.5 to 186. The mean values for MPA, MPAG and MPAG : MPA were 9.5 µg/mL, 62.3 µg/mL and 13.5, respectively. Clearly, this assay can aid clinicians develop personalized therapy strategies to avoid toxicity and maintain efficacy.
Conclusion
This method includes single dilution step, protein precipitation, ultracentrifugation and gradient chromatography. Sample preparation is rapid and efficient. This method avoids the use of more complex liquid–liquid extraction or solid-phase extraction procedure, which substantially decreases set-up time. This method has been applied to measure MPA and MPAG concentrations in plasma for pharmacokinetic studies and for monitoring clinical use of MPA prodrugs.
References
1. Bullingham RE, Nicholls AJ, Kamm BR. Clinical pharmacokinetics of mycophenolate mofetil. Clin Pharmacokin. 1998; 34: 429–455.
2. Shaw LM, Korecka M, et al. Mycophenolic acid pharmacodynamics and pharmacokinetics provide a basis for rational monitoring strategies. Am J Transplant. 2003; 3: 534–542.
3. Staatz CE, Tett SE. Clinical pharmacokinetics and pharmacodynamics of mycophenolate in solid organ transplant recipients. Clin Pharmacokinet. 2007; 46:13–58.
4. Cattaneo D, Baldelli S, Perico N. Pharmacogenetics of immunosuppressants: progress, pitfalls and promises. Am J Transplant. 2008; 8: 1374–1383.
5. Indjova D, Kassabova L, Svinarov D. Simultaneous determination of mycophenolic acid and its phenolic glucuronide in human plasma using an isocratic high-performance liquid chromatography procedure. J Chromatogr B Analyt Technol Biomed Life Sci. 2005; 817: 327–330.
6. Patel CG, Akhlaghi F. High-performance liquid chromatography method for the determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human plasma. Ther Drug Monit. 2006; 28: 116–122.
7. Bahrami G, Mohammadi B. An isocratic high performance liquid chromatographic method for quantification of mycophenolic acid and its glucuronide metabolite in human serum using liquid-liquid extraction: application to human pharmacokinetic studies. Clini Chim Acta. 2006; 370: 185–190.
8. Mino Y, Naito T, et al. Simultaneous determination of mycophenolic acid and its glucuronides in human plasma using isocratic ion pair high-performance liquid chromatography. J Pharm Biomed Anal. 2008; 46: 603–608.
9. Watson DG, Araya FG, et al. Development of a high pressure liquid chromatography method for the determination of mycophenolic acid and its glucuronide metabolite in small volumes of plasma from paediatric patients. J Pharm Biomed Anal. 2004; 35: 87–92.
10. Westley IS, Sallustio BC, Morris RG. Validation of a high-performance liquid chromatography method for the measurement of mycophenolic acid and its glucuronide metabolites in plasma. Clin Biochem. 2005; 38: 824–829.
The authors
Catherine Misch MLT and Peter Tang* PhD
Department of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
*Corresponding author
E-mail: peter.tang@cchmc.org
March 2026
Prins Hendrikstraat 1
5611HH Eindhoven
The Netherlands
info@clinlabint.com
PanGlobal Media is not responsible for any error or omission that might occur in the electronic display of product or company data.