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Original Article

Comparison of Novel Immunoassay With Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) for Therapeutic Drug Monitoring of Clozapine

Buckley, Tiffany PharmD, BCPS*; Kitchen, Christopher MA, MS; Vyas, Gopal DO; Siegfried, Nathan A. PhD; Tefera, Eshetu MS; Chen, Shuo PhD; DiPaula, Bethany A. PharmD, BCPP*; Kelly, Deanna L. PharmD, BCPP

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doi: 10.1097/FTD.0000000000000777
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Abstract

INTRODUCTION

Therapeutic drug monitoring (TDM) is the clinical practice of measuring the blood levels of medication to achieve the target concentrations to improve efficacy, reduce adverse events, or monitor adherence.1 The goal of TDM is to use data on drug level to optimize clinical outcomes and assist with prescribing decisions.1,2 The benefits of TDM in psychiatric and neurologic treatment include medication use in vulnerable populations, those with suspected pharmacokinetic interactions, or those on medications for which therapeutic ranges are suggested.3 Nonresponse at therapeutic doses, uncertain drug adherence, suboptimal tolerability, and pharmacokinetic drug–drug interactions are typical indications for TDM and can be used to consider the interindividual variability of pharmacokinetics, thus enabling personalized pharmacotherapy.4

In 2004, the TDM task force of the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) issued their first guidelines for TDM in psychiatry,5 followed by an update in 2011.4 The most recent guidelines available in 2017 were made available with the goals to improve mental health treatment, accelerate patient recovery, and reduce health care costs.3 However, in patients with schizophrenia, lifelong antipsychotic treatment is often needed. TDM is less established than in other areas of medicine owing to variations in the level of evidence and lack of clearly defined therapeutic ranges for most antipsychotic treatments.6 Nonetheless, TDM can be used to guide treatment7 for the potent antipsychotic clozapine.

Clozapine is a second-generation antipsychotic medication considered the most effective first-line therapy for treatment-resistant schizophrenia.8–11 However, clozapine is underused owing to challenging Food and Drug Administration (FDA)-required absolute neutrophil count monitoring, side effects, need for registration in the clozapine Risk Mitigation and Evaluation Services (REMS) and other logistical limitations.7,12 Numerous clinical schizophrenia treatment guidelines, including the Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology and the Schizophrenia Patient Outcomes Research Team (PORT) recommend TDM of clozapine to guide treatment.3,13 Data suggest that to maximize efficacy, the minimal threshold concentration is approximately 350 ng/mL.3,13 If a patient fails to respond adequately to clozapine, measurement of serum clozapine level is recommended to ensure that a therapeutic threshold concentration has been achieved; if clozapine level is too low, the dose should be increased accordingly with attempts to minimize side effects.13 Because clozapine is the most effective agent in schizophrenia, removing barriers to its use and maximizing time in its therapeutic range gives challenging patients the best chance for recovery.

In addition to maximizing efficacy, monitoring serum clozapine concentrations may assist with medication adherence. Nonadherence is associated with poor outcomes, relapse, rehospitalizations, longer duration to achieve remission, and increased suicide risk.14–18 Declining serum clozapine levels when a patient becomes symptomatic can serve as an early indicator of potential nonadherence, thus prompting clinical intervention for preventing detrimental consequences.8

Furthermore, another benefit of monitoring serum clozapine levels is to closely monitor side effects and toxicity during pharmacokinetic-related changes in patients. Clozapine is metabolized via various cytochrome P450 (CYP) liver enzymes, including CYP1A2, 3A4, and 2D6, and is therefore subject to drug–drug interactions; that is, inducing or inhibiting the substances of these enzymes can increase or decrease serum clozapine concentrations.19 For example, clozapine serum levels can be lowered by 30% by the aromatic hydrocarbons produced during cigarette smoking, which could lead to relapses or toxicity during smoking changes.20,21 The risk of very high clozapine serum levels during infections have been noted, including risk for potential toxicity and up to 5-fold increase in levels.22 Notably, although a toxic threshold for clozapine levels has not been established, some side effects are serum level-dependent, and very high clozapine levels have contributed to death.23 Research is ongoing to assess the ratio of norclozapine, the active metabolite of clozapine, to clozapine as a marker for side effects and efficacy; however, this is not currently recommended in TDM.23

Unfortunately, serum clozapine levels are underutilized, despite serving as a parameter for monitoring patient adherence and identifying aberrant pharmacokinetic interactions.24 Underutilization of serum levels may be caused by various reasons, including additional requirement for venous draws, lack of education, or most commonly, the fact that results of serum level tests take several days to be retrieved, and real-time dose adjustments are often unavailable when needed.25 This impedes optimal treatment and requires a clinical team to bring the patient back for follow-up visits or the information will not be used until a later visit. These levels take a few days to be retrieved because clozapine determination is measured by liquid chromatography/tandem mass spectrometry (LC-MS/MS) or high-performance liquid chromatography-ultraviolet, for which samples are often sent offsite for analysis.

If clozapine levels were available at the point-of-care, this could significantly reduce the logistical issues arising from coordination with outside laboratories and decrease the time required for results to be retrieved.11,26 Moreover, one of the most widely identified barriers in clozapine treatment are concerns for patient refusals of blood draws,25,27–29 and point-of-care results obtained via fingerstick test could ease some of these burdens. Immunoassay is a test method that can be developed and used in point-of-care devices. A novel immunoassay test, the first FDA approved de novo Clozapine Test System, (MyCare Psychiatry Clozapine Assay Kit, Saladax Biomedical Inc, Bethlehem, PA) allows miniaturization of the equipment needed to perform testing. This immunoassay is a homogenous 2-reagent nanoparticle agglutination assay that can detect clozapine levels in human serum.30 Measurements are performed by clinical chemistry analyzers that spectrophotometrically detect changes in absorbance, which reflect the concentration-dependent formation of aggregates via drug and drug-conjugates binding to drug-specific antibodies that are covalently bound to nanoparticles.30

For TDM to be successful, the process of level quantification should be validated using real-world samples to ensure that the results are accurate, precise, selective, and sensitive.3 Clozapine immunoassay has been analytically validated, and high correlations between immunoassay technology and LC-MS/MS have been shown using 213 samples.31 However, the testing had not been studied in patients not treated with clozapine or in healthy control participants. Thus, we evaluated clozapine level determination in 117 participants by sending serum to both a national reference laboratory (NRL) that used LC-MS/MS and to a site that used a novel immunoassay for clozapine level determination.

METHODS

Participants

We recruited 117 participants, including those with schizophrenia on clozapine, participants with schizophrenia not on clozapine, and healthy controls. All participants were recruited at the Maryland Psychiatric Research Center in Baltimore, MD. All arrived for a single blood draw assessment and clinical interview. The study was approved by the University of Maryland Institutional Review Board, and all patients signed informed consent before participation.

Inclusion and Exclusion Criteria

Participants aged 18–65-years-old and of all sexes, races, and ethnicities were eligible to participate in the study. Inclusion and exclusion criteria were met for 3 groups; those with a diagnosis of schizophrenia and receiving clozapine (N = 45), those with a diagnosis of schizophrenia and not receiving clozapine (N = 24), and a third group of healthy control participants (N = 45) with no major psychiatric diagnosis and no antipsychotic or clozapine use. Participants with schizophrenia met the Diagnostic and Statistical Manual of Mental Disorders-Text Revised, Fourth Edition (DSM-IV-TR).32 If a participant with schizophrenia was receiving clozapine or another antipsychotic, he/she was required to be on the same antipsychotic medication for at least 2 months. Participants were excluded from the study if they lacked the ability to give consent, which was defined as a score of 10 or less on the twelve-point Evaluation to Sign Consent.33

Study Procedure

Between April 2015 and August 2016, participants (n = 117) enrolled in a one-time cross-sectional study consisting of a single blood draw. At the time of the blood draw, demographic and clinical information was gathered, including age, sex, race-ethnicity, psychiatric diagnoses, medications, tobacco/nicotine use, and caffeine use. A complete metabolic panel (Chemistry 14 panel) and ascorbic acid levels were analyzed by an NRL in the United States for all participants.

One whole blood sample was collected, processed, and centrifuged, and the serum was aliquoted into approximately 3 freezer tubes. One aliquot was sent to the NRL for standard processing by LC-MS/MS for clozapine, norclozapine, and total clozapine levels, and the results were retrieved. A second frozen aliquot was sent to Saladax Biomedical for serum clozapine level determination by the MyCare Psychiatry Clozapine Assay Kit, (Saladax, Bethlehem, PA, USA), which uses an immunoassay technology to measure serum clozapine levels using automated clinical chemistry analyzers. Samples were analyzed on 3 occasions on 2 different Beckman AU480 clinical chemistry analyzers (Beckman Coulter, Brea, CA, USA). Before any participant serum sample was analyzed, each analyzer underwent a six-point calibration that was validated by measuring 3 levels of controls. In the morning, samples were analyzed on one analyzer. In the afternoon, samples that were positive for clozapine were then re-analyzed on the original analyzer and on an additional analyzer. A third frozen aliquot was sent in January of 2019 to the same NRL for a second assessment of clozapine by LC-MS/MS. It is believed that freezing should not affect clozapine level determination, because previous data suggested stable clozapine levels after freezing.34

Characteristics of LC-MS/MS and Immunoassay Technology

LC-MS/MS and a novel immunoassay technology were utilized to determine clozapine levels. The NRL was included to be used as an assay in the institutional review board-approved protocol to be a testing standard, but was not involved in the study design or process. Saladax did participate, knowing the study procedures; however, the protocol was written and all data were collected a few years before their involvement.

According to the NRL, the LC-MS/MS assay was tested against over 100 common drugs, vitamins, and endogenous compounds. No interfering substances were denoted. The lower limit of quantitation of clozapine by LC-MS/MS was 20 ng/mL with an upper limit of quantitation of 1000 ng/mL. The assay was validated with up to a 4-fold dilution factor, making the upper limit of the reportable range to be 4000 ng/mL. Measurement uncertainty with quality control level 1 analytical measurement uncertainty was 81.30 ± 15.88 ng/mL, and that with quality control level 2 analytical measurement uncertainty was 348.3 ± 68.2 ng/mL. The inter-run coefficient of variation was less than 11% at 2 different levels, and the intra-run coefficient of variation was less than 7% at 2 different levels of clozapine. The NRL participates biannually in the College of American Pathologists external proficiency testing survey for TDM of clozapine.

For the immunoassay validation, 191 compounds including first- and second-generation antipsychotics, commonly co-administered medications, frequently used medicines both prescribed and over-the-counter were tested for cross-reactivity in the immunoassay. None of them caused a clinically significant bias (<10%) in the results. Detection limits were established using Clinical and Laboratory Standards Institute (CLSI) guideline EP17-A2.35 The limit of quantitation was 68 ng/mL, and the limit of detection was 39 ng/mL. Precision was evaluated according to CLSI Guideline EP05-A3.36 Within-run precision with clinical samples, spiked serums, and controls was 3.3%–4.6%. The within-laboratory coefficient of variation was 4.0%–7.8%. Linearity was evaluated according to CLSI guideline EP6-A.37 The assay was linear from 68 to 1500 ng/mL, with deviation from linearity for 11 concentrations of ≤10%.

Statistical Analysis

Demographic information was reported descriptively. Pearson correlation coefficients were used to examine the relationship between the serum clozapine levels determined by LC-MS/MS and those determined by immunoassay. A linear mixed-effects model was used to examine the similarity of samples, and linear regression analysis was performed to examine the impact of age, sex, race-ethnicity, smoking status, total protein, globulin, albumin, and ascorbic acid on serum clozapine levels determined by both assays. Concordance correlation coefficient (CCC) was used to measure the agreement between measurements at different temporal points for each of the 2 laboratory assay techniques, 2 separate samples for the NRL, and 3 runs on 2 different machines for the immunoassay. Data were reported as mean ± SD. A significant finding was defined by a P-value <0.05.38

RESULTS

Overall, 117 participants were enrolled in the study. The demographic and baseline clinical information for the participants with schizophrenia on clozapine (N = 48), participants with schizophrenia not on clozapine (N = 24), and healthy controls (N = 45) is shown in Table 1.

TABLE 1. - Demographic and Baseline Clinical Information of all Participants
Variable Overall Group (n = 117) Patients With Schizophrenia Treated With Clozapine (n = 48) Patients With Schizophrenia Not Treated With Clozapine (n = 24) Healthy Controls (n = 45)
Age, yr, mean ± SD, range 40.26 ± 13.24 (19–64) 41.55 ± 13.17 (19–61) 44.63 ± 10.87 (23–63) 36.24 ± 13.59 (20–64)
Male, n (%) 72 (61.54%) 33 (68.75%) 14 (58.33%) 25 (55.56%)
Female, n (%) 45 (38.46%) 15 (31.25%) 10 (41.67%) 20 (44.44%)
African American, n (%) 55 (47.00%) 17 (35.42%) 18 (75.00%) 20 (44.44%)
Caucasian, n (%) 49 (41.88%) 28 (58.33%) 4 (16.67%) 17 (37.78%)
Other races, n (%) 13 (11.11%) 3 (6.25%) 2 (8.33%) 8 (17.78%)
Smokers, n (%) 48 (41.03%) 24 (50.00%) 17 (70.83%) 7 (15.50%)
Non-clozapine antipsychotic, n (%) 42 (36.21%) 18 (38.30%) 24 (100%) 0 (0%)
Total protein, g/mL, mean ± SD, range 7.01 ± 0.47 (5.9–8.3) 6.84 ± 0.37 (6.0–7.5) 6.91 ± 0.49 (6.2–8.2) 7.25 ± 0.47 (5.9–8.3)
Albumin, g/dL, mean ± SD, range 4.46 ± 0.30 (3.7–5.3) 4.45 ± 0.28 (3.8–4.9) 4.36 ± 0.29 (3.8–5) 4.53 ± 0.32 (3.7–5.2)
Globulin, g/dL, mean ± SD, range 2.55 ± 0.42 (1.6–4) 2.39 ± 0.33 (1.6–3.1) 2.55 ± 0.45 (1.8–3.9) 2.72 ± 0.43 (2–4)

Clozapine Level Determinations

For participants with schizophrenia on clozapine, the mean clozapine level determined by LC-MS/MS was 16.2% lower (414.98 ± 186.29 ng/mL) than that determined by immunoassay (482.08 ± 270.88 ng/mL) (P = 0.013). However, there was a strong correlation between the clozapine levels determined by LC-MS/MS and those determined by all 3 immunoassays (r = 0.84, P-value < 0.001). Based on regression analysis, each unit increase in the clozapine level determined by the NRL predicted a 1.27-unit higher immunoassay level.

Three of 24 clozapine levels (12.5%) determined by LC-MS/MS for participants with schizophrenia not on clozapine tested positive for clozapine (the mean clozapine level in the non-clozapine group was 30.33 ± 16.17 ng/mL, range 21–49 ng/mL). For participants with schizophrenia not on clozapine, the immunoassay technology produced no false positives. Likewise, 15/45 false positives (33.33%) were obtained by LC-MS/MS (mean 44.8 ± 34.26 ng/mL, range 21–159 ng/mL) for healthy controls, but none were reported with immunoassay.

We also assessed the within-lab agreement in clozapine level determination, and found that the agreement for the NRL was CCC = 0.87, 95% confidence interval = 0.690–0.970, whereas the immunoassay agreement on repeat testing yielded CCC = 0.99, 95% confidence interval = 0.979–0.997. This showed the superiority of the immunoassay over the NRL in agreement for repeat testing.

Impact of Demographic and Baseline Clinical Information on Clozapine Levels

Linear regression analysis was performed to assess the significance of demographic and baseline clinical information on clozapine levels determined by both assays. For LC-MS/MS, the linear regression analysis found no significant impact for age (P = 0.44), sex (P = 0.35), tobacco use status (P = 0.71), albumin (P = 0.21), globulin (P = 0.24), and ascorbic acid levels (P = 0.10). However, there was a significant association between African Americans and clozapine levels. The mean clozapine values were predicted to be 113.38 units higher in African Americans than in non-African American participants (P = 0.045). There was also a significant relationship between serum total protein levels and clozapine levels, with each unit in total protein predicting a 145.6-unit increase in clozapine level on LC-MS/MS.

Clozapine measurement with immunoassay showed no significant impact for age (P = 0.76), sex (P = 0.097), tobacco use status (P = 0.68), albumin (P = 0.06), globulin (P = 0.45), and ascorbic acid levels (P = 0.055). However, as with LC-MS/MS, there were significant associations between clozapine levels, race, and serum protein levels. In African Americans, the level predicted by immunoassay was 190.8 units higher than that in non-African American participants (P = 0.02). Moreover, as with LC-MS/MS, each unit increase in serum total protein levels predicted a 225.5-unit increase in clozapine levels determined by immunoassay (P = 0.036).

DISCUSSION

We reported that the novel immunoassay technology for clozapine level determination was highly correlated to LC-MS/MS and produced no false-positive results, as observed with LC-MS/MS in 26% of all participants not treated with clozapine. The mean clozapine levels were higher with immunoassay than with LC-MS/MS, but this 16.2% difference falls within acceptable standards and would be unlikely to affect patient management when clozapine blood levels are used in conjunction with other clinical information and previous values. In addition, the repeat agreement values with immunoassay were significantly better than that with the NRL, showing that between-run reproducibility and agreement was significantly better with immunoassay than with the NRL.

In addition, the lack of false-positive clozapine values obtained using immunoassay may indicate that the immunoassay had a higher specificity for clozapine than that of LC-MS/MS conducted by the NRL. The 18 false positives obtained by LC-MS/MS showed relatively low clozapine values (mean 42.39 ± 32.06, range 21–159 ng/mL). Eleven patients (2 participants not on clozapine; 9 healthy controls) with false-positive values for clozapine exhibited levels lower than 39 ng/mL, which would likely be clinically insignificant. Although LC-MS/MS is considered the gold standard, there is still a possibility of error with all types of measurement. We are unsure of the explanation of the high rates of false positive clozapine values as observed with LC-MS/MS. These finding may be because of poor laboratory techniques at the NRL, interference, contamination or carry-over. The laboratory reported interference testing for over 100 medications with LC-MS/MS; thus, it appeared that the likelihood of interference from other compounds was very low. Owing to the wide variability in co-medications, the number of samples within groups was too small for running correlations. However, upon visual inspection, there appeared to be no trends in co-medication use contributing to the false positives. We have kept the NRL laboratory in this manuscript unnamed, but we suggest that these LC-MS/MS value errors could be present at any facility performing LC-MS/MS. A search of PubMed and EMBASE yielded no documented cases of false positive values for clozapine with LC-MS/MS. However, the literature does provide a case of a false-positive clozapine result determined by HPLC with ultraviolet detection.39 This anomaly was discovered accidently owing to a clerical error and was verified later by LC/MS–MS.39 This study highlighted that the possibility of false positives cannot be discounted and is an area that warrants further exploration. Based on these findings, our data suggested that immunoassay may be superior to LC-MS/MS in reproducibility and accuracy (no false positives) for the determination of clozapine levels in patient care; however, further studies are required to identify what is considered the gold standard now that FDA has approved the first de novo Clozapine Test System for clozapine measurement in human specimens.

We found that variations in clozapine level were not correlated to age and sex, but did differ according to ethnicity and were influenced by total protein serum levels in both immunoassay and LC-MS/MS assay. It is interesting that the total protein level was correlated to clozapine values in both assays. We do know that clozapine is highly protein-bound; however, standard guidance or treatment guidelines for clozapine serum concentrations do not routinely take total protein levels into account. Although there are many differences in LC-MS/MS extraction protocols that could affect the recovery of protein-interacting clozapine, which may contribute to inter-lab variability, we would expect that both immunoassay and LC-MS/MS assay to similarly detect both bound and unbound clozapine. It is also known that clozapine levels increase during acute infections, but it remains unknown whether this is because of inflammatory markers and downregulation of CYP enzyme activity or to increased total protein concentration,40 which would also indicate that total protein values are important for clozapine dosing and targeting of blood clozapine level. More studies are needed to elucidate how to account for total protein values in accurate determination of clozapine values and in clinical decision-making.

Finally, although clozapine levels are used for dose optimization as recommended by published guidelines, the use of the clozapine:norclozapine (CLZ:NOR) ratio is not established. The CLZ:NOR ratio may be useful for obtaining information on recent adherence, although it is not necessary for routine patient monitoring nor found to be a good measurement of clozapine response and is not included in the 2017 update to the Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology for testing of blood clozapine level.3,41 However, it is still noteworthy that the immunoassay only measures clozapine values, and not norclozapine This could be a limitation in situations when a clinician feels that the CLZ:NOR ratio could help guide treatment, such as in predicting cardiometabolic outcomes.42 Likewise, norclozapine levels may be useful to help explain abnormal levels in some patients, which may be due to the use of inhibiting medications or rapid metabolism.43 In addition, Figure 1 showed that in general, there was great variability in levels, which can be explained by genetic and metabolic differences, interacting compounds, dose, and other potential interferences not explored in this study.

FIGURE 1.
FIGURE 1.:
Correlation between 3 time average CLZ immunoassay to the National Reference Laboratory.

CONCLUSIONS

Our results suggested that routine LC-MS/MS analysis of clozapine levels may have some challenges, such as low reproducibility of values and possibility for false positives, as we found that this assay produced a false positive for 26% of all participants not treated with clozapine. Our findings also suggested that the use of a new immunoassay technology was very promising for clinical use in the determination of clozapine levels. Because immunoassays can be adapted for use in point-of-care devices,44 clozapine testing at the point-of-care could be feasible and aid in improving the logical blood draw barriers that currently exist. The next step for validation of point-of-care testing would be to ensure that whole blood capillary values with clozapine would be similar to serum values, as some have suggested45; however, fingerstick testing would be needed for verification. Moreover, further research is needed to elucidate the role of total protein values in clozapine blood level determination, because low total protein concentrations would likely result in low clozapine values. In conclusion, immunoassay determination appeared very promising, and point-of-care development of a fingerstick test based on this technology could revolutionize treatment with more optimized care for people treated with clozapine.26

ACKNOWLEDGMENTS

The authors would like to acknowledge the help of Vincent Happ, Jodi Courtney and Karen Horn in the preparation of this manuscript. This study was funded in part by NIMH gratn R01 MH105571-01.

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Keywords:

clozapine level; immunoassay; therapeutic drug monitoring; point-of-care device; schizophrenia

Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.