Therapeutic drug monitoring of levetiracetam: Method validation using high-performance liquid chromatography-ultraviolet detector technique and usefulness in patient care settingRP Munshi, JV Vishwakarma, NR Gawde
Department of Clinical Pharmacology, TN Medical College and BYL Nair Charitable Hospital, Mumbai, Maharashtra, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/jpgm.jpgm_467_21
Source of Support: None, Conflict of Interest: None
Keywords: Antiepileptic drugs, compliance, drug–drug interactions, HPLC-UV, seizure control
Levetiracetam is a novel broad-spectrum anticonvulsant agent with favorable pharmacokinetics, unique safety, and minimal drug–drug interaction profile. Its pharmacokinetics are linear with low inter-subject and intra-subject variability. It does not significantly bind to serum proteins signifying least competition for protein binding with other antiepileptic drugs, viz., sodium valproate, phenytoin, phenobarbitone, carbamazepine, oxcarbazepine, lacosamide, lamotrigine, clobazam, topiramate, zonisamide, and lithium. It is recommended as add-on medication for the treatment of partial seizures, myoclonic seizures, and primary generalized seizures in adults and as add-on therapy for children aged 1month and older with focal seizures, children aged 12 years and older with myoclonic seizures from juvenile myoclonic epilepsy, and children aged 6 years and older with generalized tonic-clonic epilepsy.
Although levetiracetam has excellent oral absorption and bioavailability (>95%) making for easy dosing and tolerability, it is primarily excreted unchanged in urine and hence dosage adjustments are necessary for patients with moderate-to-severe renal impairment. Thus, therapeutic drug monitoring of levetiracetam may be indicated in certain situations, such as suspicion of poor compliance, pregnancy, dosing in children and elderly and those with renal impairment, etc.,
Various chromatographic methods for the detection and quantification of levetiracetam in biological fluids have been reported. These methods include high-performance liquid chromatography (HPLC), HPLC methods coupled with ultra-violet (UV) or diode array detection, liquid chromatography coupled to mass spectrometry (LC/MS), and gas chromatography (GC) with various detection systems. Some of these procedures are complicated, time consuming, expensive, and lack selectivity.,,,,
Being a public sector tertiary care municipal hospital with limited resources, this study aimed to develop a rapid, simple, and validated HPLC-UV method for effective measurement of serum levetiracetam levels that would assist in determining drug compliance, drug–drug interactions, and clinical benefits in epileptic patients.
The study [EC Project No. ECARP/2020/198] was approved by the Institutional Ethics Committee with a waiver of informed consent. The study was conducted in compliance with the updated Standards for Reporting Diagnostic Accuracy Studies reporting guidelines and Indian Good Clinical Practice Guidelines. The study involved analysis of patient blood samples and followed ICMR Ethical Guidelines for Biomedical Research on Human Participants during the study.
Method development and validation for estimation of levetiracetam level using HPLC-UV technique
]The reported TR for levetiracetam is 5–41 μg/ml. We developed our method for the serum estimation of levetiracetam modifying HPLC techniques mentioned in literature.,,,, Liquid–liquid phase extraction method was used to prepare the samples prior to the running on the analytic HPLC-UV system. Theophylline was employed as the internal standard (IS) to control the extraction procedure. Levetiracetam pure powder was received from Sun Pharmaceuticals (Mumbai, India), whereas theophylline was purchased from Sigma-Aldrich (Mumbai, India). Other chemicals and reagents were purchased from Merck and Sigma Aldrich. Sample separation was carried out on Reverse Phase HPLC (Thermo-Finnigan) Surveyor with UV Detector using Chromquest software.
The mobile phase consisting of potassium dihydrogen phosphate buffer and acetonitrile was filtered through 0.47 μ membrane filter, degassed, and then pumped into the column at a flow rate of 1.2 ml/min. The column oven temperature was maintained at 40°C. The detection was monitored at 205 nm and the run time was 15 mins. The volume of injection loop was 10 μL. Stock solutions of levetiracetam and theophylline were prepared by adding 10 mg of the drug to 10 ml methanol as diluent to get a concentration of 1 mg/ml. About 200 μL of serum sample (calibration standards, quality control samples, and patient samples) was spiked with 10 μL of IS and vortexed. About 200 μL of dimethyl sulfoxide (DMSO) was added as an extracting agent. The mixture was vortexed for 30 s and then centrifuged at 12,000 rpm for 10 mins at 21°C. The supernatant was carefully separated to HPLC-UV vials.
The developed method was validated according to the International Conference on Harmonization (ICH) Q2 (R1) 2005 updated guidelines using the six parameters of specificity, precision, accuracy, linearity, limit of detection (LOD) and limit of quantification (LOQ), and robustness.
Secondary data analysis to assess the usefulness of serum levetiracetam estimation in clinical setting
Estimation of serum levetiracetam levels from blood samples of epileptic patients attending the TDM OPD of the Department of Clinical Pharmacology of our hospital was initiated using the method mentioned hereinabove in 2015 as a part of the patient care services with administrative approval. In late 2020, we decided to analyze the data generated in 6 years for levetiracetam to assess the usefulness of the assay method with respect to the clinical response. The clinical details of all patients, viz., age, gender, probable diagnosis, investigation reports, such as EEG, CT, and MRI, details of seizure control including number of breakthrough seizures, indication for TDM, AED details, such as dosage, frequency, any alterations, co-morbid conditions, concomitant medications, previous TDM reports, etc., documented in a specific proforma for the TDM OPD were accessible following Ethics Committee approval for waiver of informed consent. Using these proformas, analysis of estimated levels of levetiracetam was carried out with respect to TR reported in the literature, age, changes in pregnancy, details of seizure control including number of breakthrough seizures during the last 6 months, mono or polytherapy with antiepileptic drugs, drug compliance, and adverse drug reactions (ADRs) reported by the patients. For the purpose of the study, patients were considered to have good seizure control if they had at least a 50% reduction in the number of seizures during the past 6 months and good drug compliance if they consumed at least 70% of their medicines per month.
Descriptive statistics was used for analysis. Categorical data (proportion of patients by age and levetiracetam levels as per the TR) were expressed as percentages. Analysis was done using Microsoft Excel 2010.
Method development and validation for estimation of levetiracetam level using HPLC-UV technique
The modified method to estimate levetiracetam levels using the HPLC-UV technique was developed and validation parameters were as follows:
Secondary data analysis to assess the usefulness of serum levetiracetam estimation in clinical setting
From 2015 to 2020, 1383 patients receiving levetiracetam underwent TDM using modified assay method. Levetiracetam levels were analyzed on the basis of TR, age [Table 3], mono or polytherapy, adherence to therapy, number of breakthrough seizures, and reported ADRs. It was found that 1107 (80.04%) patients had levetiracetam levels within the TR (5–41 μg/ml), 44 (3.18%) patients greater than TR ranging from 43 to 51 μg/ml, and 232 (16.78%) patients less than TR, when consumed either alone or with other antiepileptic co-medications.
Of 1383 patients, 635 were children and adolescents (less than 18 years), 683 were adults (18 to less than 65 years), 25 were pregnant women, and 40 belonged to the elderly group (>65 years). Age-wise analysis of levetiracetam levels showed that of 683 adults, 543 (79.50%) patients had serum levetiracetam levels within TR, 116 (16.98%) had levels below, and 24 (3.51%) patients had levels greater than TR. Of these 683 adults, 461 (67.50%) patients received levetiracetam as add-on therapy, whereas 222 (32.50%) patients received levetiracetam as monotherapy. Ninety-seven patients on polytherapy had levels less than TR (21%) compared to 19 patients on monotherapy (8.56%).
Poor compliance was observed in 128 patients (18.574%), whereas poor seizure control was reported in 179 adults (26.21%), of whom 141 (78.78%) were on polytherapy [Table 4].
In case of 635 children, 520 (81.89%) children had serum levetiracetam levels within TR. Of these, 429 patients exhibited good seizure control. The levels were found to be less than the TR in 95 (14.96%) and greater than the TR in 20 (3.15%). Of 635 children, 168 (26.41%) patients received levetiracetam as an add-on therapy, whereas 467 (73.58%) patients received levetiracetam as monotherapy. About 431 patients on monotherapy had levels within TR (92.29%) compared to 89 patients on polytherapy (53%). Poor compliance was observed in 226 patients (35.59%). Poor seizure control was reported in 157 children, 82 of whom were on polytherapy [Table 4].
Of 40 elderly patients, five patients exhibited serum levetiracetam values less than TR (three on monotherapy and two on polytherapy), whereas 35 patients had levetiracetam values within TR (25 on monotherapy and 10 on polytherapy). However, all these patients had good seizure control on levetiracetam at a dose of 500–1,000 mg/day, although poor compliance was observed in two patients with low-levetiracetam levels. Elderly patients receiving levetiracetam as monotherapy were 28 (70%), whereas 12 (30%) were consuming levetiracetam as add-on therapy [Table 4].
Compliance to therapy was assessed by medical history during consultation. Poor compliance was noted in 112 patients (48.28%; 27 on monotherapy and 85 on polytherapy) with low-levetiracetam levels, 160 patients (14.45%; 67 on monotherapy and 93 on polytherapy) with levels within TR, and 15 patients (34%; 4 on monotherapy and 11 on polytherapy) with levels greater than TR.
Assessment of the seizure control with levetiracetam showed that 913 of 1107 patients (82.47%) whose levetiracetam levels were within TR had good seizure control with fewer breakthrough seizures compared to previous records (from 10 to 12 seizures in previous 6 months to three to four seizures in the next 6 months once started on levetiracetam). However, 194 patients (103 adults and 91 children) with levels within TR did not report a decrease in the number of breakthrough seizures [Table 4].
In the case of 232 patients in whom levetiracetam levels were less than TR, poor compliance was documented as the reason for low- or non-detectable levetiracetam values in 112 (48.28%) patients, of whom 85 patients were receiving multiple AEDs. In 136 (58.62%) patients with low-drug levels (111 of whom were on polytherapy), an increase in seizure frequency from 3–4 to 6–8 seizures during a period of 6 months was reported. Based on the TDM reports, the clinicians adjusted the treatment regimen in 120 (88.24%) patients by increasing the dose of levetiracetam in 48 patients, adding clobazam to the drug regimen in 35 patients or adding another anti-epileptic drug in 37 patients for better seizure control.
In 44 patients in whom serum levetiracetam levels were greater than TR, detailed history taking provided plausible explanation for high levels in 15 patients, viz., that four patients had consumed the medication only a few hours before the test, the drug had been administered as an intravenous (IV) medication and blood for TDM had been collected from the same vein in six patients, and recent increase in dosage in five patients [from 750 to 1000 mg (n = 2) and 1000 to 1500 mg (n = 3)]. Twenty-nine of the 44 patients were on polytherapy. Fifteen (34.09%) of 44 patients with high-levetiracetam levels complained of giddiness, headache, tiredness, and irritability; 11 (25%) complained of an increase in the seizure frequency during the past 6 months along with adverse reactions in eight of them because of which the clinician/s lowered the dose of levetiracetam.
There were 25 pregnant patients in the adult cohort whose samples were sent for TDM in the third trimester. All these pregnant women were on levetiracetam monotherapy. Of these, nine (36%) patients receiving 750 mg dose per day of levetiracetam had values within the TR although towards the lower limit of the TR, whereas 16 (64%) of the pregnant women consuming 500 mg dose per day levetiracetam had values less than the TR. Five of these women reported breakthrough seizures during pregnancy and their dose was increased by 500 mg by the clinician. Clobazam was added in another seven women with low-levetiracetam levels for better seizure control, whereas the same dose was maintained in four women with low levels.
Of the 641 patients who also received other antiepileptic drugs as concomitant medications, 446 (69.58%) patients had levetiracetam values within TR, whereas 29 (4.52%) patients had values above and 166 (25.9%) patients had values less than TR. Potential drug–drug interaction/s that might have affected levetiracetam's serum concentration when given in combination with other antiepileptic drugs, viz., phenytoin, phenobarbitone, carbamazepine, oxcarbazepine, valproate, lacosamide (Lacos), lamotrigine (Lam), lithium (Li), clobazam, topiramate, and zonisamide was assessed in these patients. Twenty-nine patients on polytherapy had serum levetiracetam levels greater than TR (17 receiving single AED, nine receiving two AEDs, and three receiving three AEDs in addition to levetiracetam), whereas 166 patients on polytherapy had serum levetiracetam levels less than TR (122 receiving single AED, 38 receiving two AEDs, and six receiving three AEDs in addition to levetiracetam). Of these, 85 reported poor compliance. The common anti-epileptic drugs that affected levetiracetam levels when given concomitantly were sodium valproate, phenytoin, and carbamazepine [Figure 2] and [Figure 3].
ADRs were reported by 203 patients that included 162 patients (14.63%) with levels within TR (restlessness, generalized body ache, and excessive sleepiness). As mentioned earlier, from those having levetiracetam levels greater than TR, 15 patients (34.1%) exhibited ADRs. ADRs, such as nausea, irritability, and tiredness were also reported by 26 patients (11.21%) with low serum levels. Forty-six of 203 patients (22.66%) confirmed that they had lowered the dose on their own because of these adverse reactions. Clinicians decreased the dose in eight (0.58%) patients.
The HPLC-UV method developed in this study is simple, rapid, efficient, and reliable with a run time of 15 mins, and the whole process from sample extraction to injecting the sample in the HPLC-UV system and evaluating the chromatogram to determine the levetiracetam levels in the sample requires 30 mins.
The different chromatographic methods used to estimate serum levetiracetam (HPLC/LC-MS/GC, etc.) are mainly based on the conventional sample preparation procedures, such as liquid–liquid extraction (LLE), solid-phase extraction (SPE), plasmatic protein precipitation (PPP), and time-consuming procedure of sample extraction by nitrogen evaporation. They do have their own difficulties. For example, PPP procedure can result in signal suppression when LC/MS is used because of the presence of a precipitation agent, coprecipitation of interfering species, and lack of selectivity; SPE procedure gives interference, bad recovery with high standard deviation, is complicated, time consuming, and expensive; cartridges used for the extraction are relatively expensive with the risk of carryover and cross-contamination; and most of the chromatography techniques have tedious and time-consuming sample preparation procedures.,,,,
We made following modifications in our HPLC method compared to available literature to improve the runtime and make it more cost-effective, viz., (1) change in the ratio of the mobile phase from 94:6, v/v to 95:5, v/v; (2) change in the flow rate from 1.5 to 1.2 ml/min; (3) modification in the sample preparation procedure wherein theophylline was used as the IS instead of fluconazole and gabapentin, which are more expensive; (4) DMSO was used as the extracting agent instead of methanol; and (5) the mixture was vortexed for 30 s and then centrifuged at 12,000 rpm for 10 mins at 21°C instead of 14,000 rpm for 20 mins.
Thus, compared to the chromatographic techniques mentioned hereinabove, our method simplifies sample purification by cutting down on laborious and expensive SPE or LLE and the time-consuming step of nitrogen evaporation. This method requires a minimal sample pre-treatment that allows a large series of patient samples to be processed in a short time, which is advantageous in a hospital setting. Furthermore, the simple reversed-phase HPLC-UV chromatographic method can be adopted even by laboratories lacking sophisticated analytical equipment.
Method validation is a necessary procedure used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation are used to judge the quality, reliability, and consistency of the analytical procedure., Hence, method used in this study has been validated as per latest guidelines prior to use in clinical practice. A comparison of the results of this study method with those obtained from HPLC-UV, GC-MS, HPLC-ESI MS/MS, and UHPLC-PDA showed that there was no significant difference in the accuracy 105.02–110.6% and precision was <3.5%. The only difference was in the LOQ. It was 1.15 μg/ml of serum with the modified method and was higher than that seen with other HPLC-UV methods (1 μg/ml of plasma).,
Data analysis of 1383 patients' serum samples that were processed for the estimation of levetiracetam drug levels using this study method during the last 6 years showed that 80.04% (1107) of the patients had levetiracetam values within the TR, thus confirming levetiracetam as a preferred antiepileptic drug because of its favorable pharmacokinetics – rapid absorption, high oral bioavailability that is unaffected by food, primarily renal elimination, and a lack of CYP enzyme-inducing potential resulting in fewer alterations in the kinetics of concomitantly administered drugs.,
Striano et al. reported that levetiracetam administered at high doses (1500 mg twice a day) shows an extremely large variability in plasma concentrations with very high peak levels seen 2 hours after drug intake. This was possibly one of the reasons for values greater than the TR seen in 15 of 44 patients who came for testing only a few hours after consuming levetiracetam, or too early following a dose increase that did not allow for steady state level of levetiracetam to be achieved. In six of them, levetiracetam had been administered as an IV medication and blood for TDM had been collected from the same vein.
The authors found that 29 patients with high-levetiracetam levels were receiving polytherapy. Of these 22 were prescribed sodium valproate, a known CYP enzyme inhibitor, as a concomitant AED. Although some studies reported no pharmacokinetic interaction between sodium valproate and levetiracetam, recent studies observed that levetiracetam trough concentrations were 16% higher when co-administered with sodium valproate compared to levetiracetam monotherapy and that patients receiving sodium valproate tended to have a slightly lower clearance rate for levetiracetam, thus, increasing its serum level.,
Poor compliance was observed in 112 patients with low-levetiracetam levels, and one of the reasons given by the patients was non-affordability of levetiracetam as it is a relatively costly drug compared to the older AEDs and is not available free of cost at the hospital pharmacy it means that patients had to purchase the drug themselves. Drug–drug interactions were another possible reason for levetiracetam level being less than TR as 85 (37%) patients in this group were receiving polytherapy, especially phenytoin and carbamazepine, known CYP enzyme inducers, as concomitant AEDs. Studies have shown that patients receiving levetiracetam together with enzyme inducers exhibited a 1.3-fold higher levetiracetam clearance, thus, mandating a need to monitor levels and adjust doses in such cases.,
Poor compliance was also noted in 160 patients with levels within TR and 15 patients with levels greater than TR. Reasons elicited from the patients for not taking their medications was non-affordability in some cases, ADRs in others because of which 27 patients (12 from within TR and 15 from greater than TR) lowered the dose themselves, and lastly, the absence of seizures over few months that made the patients skip or lower their daily dose.
Clinical assessment of the seizure control with levetiracetam showed that majority of the patients responded well to levetiracetam with an overall good seizure control of 70% (1042 patients). About 913 patients with drug levels within TR had fewer breakthrough seizures compared to patients with drug levels less than TR (n = 136) and greater than TR (n = 11) who reported poor seizure control with one to two breakthrough seizures occurring every month. Patients on monotherapy had better seizure control, indicating that levetiracetam was effective in controlling seizures when given as a single AED.
Paediatric patients too responded well to levetiracetam as mentioned in the results. Polytherapy, poor compliance, and a higher clearance rate as reported in young children, especially those between 6 and 12 years, are some of the reasons for the low-levetiracetam levels observed in 95 children in this study.
All 40 elderly patients on levetiracetam in this study were well maintained on levetiracetam at a dose of 500–1000 mg/day compared to the dose range in adults of 1000–3000 mg/day (although five patients had levels less than TR) and had good seizure control and reported no ADRs. Studies have shown that elderly patients require a lower levetiracetam dose compared to young patients because of reduced clearance with increasing age. Additionally, 28 of these patients (70%) were consuming levetiracetam as monotherapy that lowered the risk of drug–drug interactions with other anti-epileptic drugs. Data of this study, thus, match with other studies that have reported that levetiracetam given as mono or add-on therapy is well tolerated and associated with an improvement of seizure control in elderly epileptic patients.
Pregnant women who came for estimation of levetiracetam levels (n = 25) were referred mainly in the third trimester as the clinicians wished to confirm the level and whether there would be a need to increase the dose of levetiracetam or add another AED for better seizure control during delivery as all these women were on levetiracetam monotherapy. Levetiracetam levels were either towards the lower limit of TR (n = 9) or less than TR (n = 16), which could be because of the low doses prescribed to these pregnant women. Additionally, levetiracetam levels are reported to decrease during pregnancy because of a higher clearance rate that is most significant in the first trimester but continues throughout pregnancy. Physiological changes seen in pregnancy, such as increased estrogen levels may lead to accelerated drug glucuronidation, increased peripheral hydrolysis of levetiracetam, altered gastrointestinal absorption because of gastric pH changes, decreased gastrointestinal motility or vomiting during pregnancy, etc. can all affect levetiracetam levels., Five of these women with low-levetiracetam levels reported breakthrough seizures during pregnancy. Thus, regular monitoring of serum levetiracetam levels throughout pregnancy is important for dose adjustments that are required to correct for decreased serum levels, especially in the last trimester.
Levetiracetam levels were within the TR in 446 (77.14%) of the 641 patients who were on multiple anti-epileptic drugs, including levetiracetam, indicating that levetiracetam per se does not cause any significant alterations in the kinetics of concomitantly administered drugs. These findings are similar to that reported in other studies., Its levels are, however, affected by anti-epileptic drugs that are enzyme inducers (carbamazepine, phenytoin, and phenobarbitone) or enzyme inhibitors (sodium valproate) when given concomitantly., Thus, monitoring of levetiracetam levels when these AEDs are given as concomitant medications may be warranted to prevent drug–drug interactions that may affect seizure control clinically.
Eleven (25%) patients with levetiracetam levels greater than TR and on polytherapy complained of an increase in the seizure frequency during the past 6 months with eight patients also complaining of adverse reactions. Although there is no clear explanation for this effect, reports of aggravation of seizures with high levels of levetiracetam has been reported, called as “paradoxical intoxication.” Other explanations given are natural fluctuations of the seizure frequency, progressive worsening of the underlying disease condition, intercurrent illness or other seizure-precipitating factors, or adverse pharmacodynamic effect and/or interaction.
Although only 203 (14.67%) patients clinically complained of ADRs with levetiracetam, 46 of them, on detailed enquiry, stated that they had lowered the dose on their own mainly because of ADRs that resulted in low-levetiracetam levels in 19 patients that was identified because of TDM. Hence, regular monitoring may help in the early detection and management of adverse reactions, which may be associated with drug levels and/or drug–drug interactions.
This study has its limitations. This study being a retrospective analysis, factors such as recall bias on the part of the patients with respect to compliance and seizure frequency may have confounded the results. This study captured data as and when the patients availed of the TDM services. A prospective longitudinal study with intensive monitoring and follow-up and inclusion of special populations such as those with renal impairment would provide a more accurate analysis of the importance of TDM of levetiracetam.
To summarize, this study demonstrates that although TDM of levetiracetam need not be performed routinely in clinical practice, its estimation is important in patients on polytherapy especially with AEDs that are hepatic enzyme inducers or inhibitors, such as valproate, phenytoin, and carbamazepine; patients with frequent breakthrough seizures in whom a dose adjustment depending on the serum drug levels may improve seizure control; and in pregnancy, especially in the third trimester to reduce the risk of seizures during the peripartum period and to assess compliance at regular intervals, especially as epilepsy is a chronic medical condition for which anti-epileptic drugs are prescribed for years.
We acknowledge Sun Pharmaceuticals Industries Limited, Mumbai, India, for their kind donation of the active pharmaceutical ingredient of levetiracetam. We also acknowledge Ms. Shraddha Jadhav and Ms Apurva Takke for their technical expertise in the method development procedure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]