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Rapid determination of acyclovir, its main metabolite 9‐carboxymethoxymethylguanine, ganciclovir, and penciclovir in human serum using LC–MS/MS

Abstract A novel MS‐based analytical method for simultaneous analysis of the antiviral drugs acyclovir, its metabolite 9‐carboxymethoxymethylguanine, ganciclovir, and penciclovir in human serum is described. These antiviral drugs are active against herpes virus infections. Acyclovir and penciclovir are regarded as safe and effective medicines with mild side effects such as headache and gastrointestinal discomfort, and ganciclovir is regarded as more toxic and is known to cause, for example, bone marrow suppression. Acyclovir’s main metabolite 9‐carboxymethoxymethylguanine is a presumptive neurotoxin and should be monitored in patients with impaired renal function or in cases with neurotoxic symptoms. A sample was prepared using protein precipitation with 1% formic acid in methanol containing isotopically labeled internal standard. Chromatographic separation on a biphenyl column and mass spectrometric detection were performed in multiple reaction monitoring (MRM) mode on a Xevo TQ‐S micro with ESI in positive ion mode, within 3 min. Inter‐day assay accuracies for the quality controls varied between 95 and 104% and intra‐day assay between 93 and 105%. Inter‐day and intra‐day assay imprecision for the quality controls ranged between 1.4 and 4.2% and 1.7 and 6.5% respectively. The lower limit of quantification for all four substances was 0.156 μmol/L. It is an accurate and reproducible method for therapeutic drug monitoring.

inhibits viral DNA polymerase and terminates the viral DNA chain.
GCV and PCV as well as the major part of ACV are excreted unchanged by the kidneys. Approximately 5-15% of ACV is metabolized in the liver by alcohol dehydrogenase to an ACV aldehyde and further on via aldehyde dehydrogenase to the main metabolite 9-carboxymethoxymethylguanine (CMMG) (de Miranda et al., 1982;Hellden et al., 2006).
Despite these antiviral drugs being available on the market for several years, no therapeutic levels nor a reliable concentrationtoxicity relationship has been clearly defined for either of the substances. The use of therapeutic drug monitoring (TDM), for example, for GCV, has therefore been questioned (Galar et al., 2021;Jager et al., 2016;Scott et al., 2004). Too high a dose in renal-compromised patients increases the probability of toxic drug levels, which is why dose adjustment is necessary for patients with impaired renal function to avoid the risk of accumulation resulting in toxic drug and metabolite levels. The incidence of ACV-induced nephrotoxicity is approximately 10-15% (Bean & Aeppli, 1985;Lee et al., 2018;Ryan et al., 2018) with frequent case reports every year since the early 1980s (Bach, 1987). For GCV, cases of neurotoxicity, hepatitis, and bone marrow suppression have been reported, but relatively few cases compared to the number of cases reported for ACV. It can be hypothesized that toxicity symptoms in these cases can be difficult to diagnose without TDM due to the similarity between side effects and the disease.
We have previously shown that CMMG is consistently increased in patients with ACV-induced neurotoxicity, mainly in patients with acute or chronic kidney disease (Hellden et al., 2003). Recent publications have shown the utility of TDM in CMMG in bone-marrow transplanted patients (Berry & Venkatesan, 2014;Brandariz-Nunez et al., 2021;Lindstrom et al., 2019). In Sweden, measurements of CMMG levels have been used as a marker of ACV-induced toxicity since 1994 and helped many Swedish physicians to distinguish between ACV toxicity and symptoms of viral encephalitis infections.
Even though TDM studies of GCV have provided contradictory results, (Vezina et al., 2014;Wiltshire et al., 2005) promising results have been shown in the prevention of drug toxicity as well as cases of subtherapeutic drug concentrations not only in Sweden (Martson et al., 2019;Martson et al., 2021;Peredo et al., 2015).
Previously published analytical methods for determination of ACV and its metabolite CMMG have used HPLC in reversed-phase mode for the separation followed by UV, UV-diode array, fluorescence detection, or MS (Dao et al., 2008;Darville et al., 2007;Svensson et al., 1997;Urinovska et al., 2021;Weller et al., 2009). In these publications, the most common reversed phases used for chromatographic separation have been various C8 and C18 phases with, for example, HSS T3 tri-functionally bonded C18 ligands that promote polar compound retention and are compatible with aqueous mobile phases. As these antiviral compounds are fairly polar and include an aromatic moiety, other reversed phases such as phenyl or biphenyl could produce variations in retention and selectivity compared to the traditional C8 and C18 phases. Previously, a phenyl-hexyl phase has been used for the separation of seven antiretroviral agents (Zheng et al., 2020). Because the polar nature of these compounds may result in poor retention and peak shape when analyzed in reversed-phase mode, chromatographic separation using hydrophilic interaction liquid chromatography (HILIC) has also been attempted (Brown et al., 2002).
With the introduction of UHPLC and the use of sub-2 μm particle columns, a reduction in separation time can be obtained while maintaining separation efficiency. Combining the peak capacity of the UHPLC column with the selectivity of the mass spectrometer generally results in increased sensitivity and reduced analysis time, particularly for multicompound methods. Several methods exist for the determination of ACV, GCV, and PCV in human plasma or serum, with or without simultaneous determination of their corresponding prodrugs (Heinig et al., 2011;Kasiari et al., 2008;Schimek et al., 2018;Shi et al., 2018;Xu et al., 2007;Yadav et al., 2009) but only a few methods include the measurement of CMMG (Darville et al., 2007;Svensson et al., 1997;Urinovska et al., 2021;Yang et al., 2007).
The aim of this work was to develop and validate a rapid, simple, sensitive, and robust LC-MS/MS method for the quantification of ACV, its metabolite CMMG, as well as GCV, and PCV in human serum for the purpose of monitoring antiviral drug levels and diagnose toxicity in critically ill patients.

| Standards and quality control samples
Stock solutions of 1 mg/mL of ACV, GCV, and PCV, ACV-IS, GCV-IS, and PCV-IS, respectively, were prepared in methanol with 1% (v/v) of hydrochloric acid. Stock solutions of 1 mg/mL CMMG and CMMG-IS were prepared in DMSO. Calibrators and quality controls (QCs) were made by spiking ACV, CMMG, GCV, and PCV into drug-free serum from healthy volunteers (blood donors from Linköping University Hospital). A six-point calibration curve was prepared by spiking drug-free serum with each of the analytes to a concentration of 160 μmol/L, followed by a serial dilution to obtain the concentration of 40, 10, 2.5, 0.625, and 0.156 μmol/L. QC samples were prepared by using separate stock solutions by adding the four analytes to drug-free serum to make the high-level QC at 120 μmol/L and was further diluted to obtain the mid-level QC (OC-II, 20 μmol/L) and the low-level (QC-I, 0.5 μmol/L) respectively. An internal standard working solution (5 μmol/L of ACV-IS, CMMG-IS, GCV-IS, and PCV-IS) was prepared in methanol with 1% (v/v) formic acid.

| Sample collection
Samples from patients were collected in serum tubes containing no anticoagulants and centrifuged for 10 minutes at 2,000g, after which serum was transferred to a new tube prior to analysis or storage at À20 C.

| Sample preparation
About 150 μL cold internal standard solution was added to 50 μL of the calibration standard, QC or serum sample, vortexed briefly followed by centrifugation at 15,000g for 5 min at 10 C. An amount of 100 μL of the supernatant was transferred to a 96-well plate and diluted with 100 μL of 1% formic acid in Milli-Q water followed by mixing for 10 min.

| Chromatography and instrumentation
LC-MS/MS analysis was performed on an Acquity (Waters, Milford, MA, USA) system equipped with a binary solvent manager, flowthrough needle (FTN) autosampler, and a mass spectrometer with an electrospray interface (ESI) operating in the positive ion mode, XEVO TQ-S micro. Chromatographic separation was performed at 40 C using a Kinetex biphenyl column (100 Â 2.1 mm, 100 Å pore size, 2.6 μm particle size) preceded by a pre-column, SecurityGuard™ Ultra biphenyl 2*2.1 mm, all from Phenomenex (Copenhagen, Denmark).
The autosampler purge solution was 10:90 methanol:water, the needle wash solution was 80:20 methanol:water, and the injection volume was 2 μL. The four analytes and their internal standards were separated in a gradient chromatographic run using a mobile phase consisting of 10 mmol/L ammonium acetate pH 6.8 (A) and methanol (B) at a flow rate of 0.5 mL/min over the total run time of 3 min. The following mobile phase gradient was used: phase B was started at 2% for 0.3 min, then increased to 25% from 0.3 to 2.0 min, further increased to 75% from 2.0 to 2.5 min, after which it was returned to the initial composition and equilibrated until 3.0 min.
The mass spectrometer was operated in positive ion mode. The instrument conditions were as follows: capillary voltage, 1.3 kV; desolvation gas flow and temperature, 1000 L/h and 500 C respectively; the source temperature, 150 C; and the sample cone gas flow, 50 L/h.
All analytes and internal standards were detected using multiple reaction monitoring (MRM) recording one quantifier ion, one qualifier ion for each of the analytes, and a quantifier ion for the internal standards. The specific mass spectrometric settings for each compound are presented in Table 1. Six-level calibration was performed before every analysis, and concentrations were calculated using TargetLynx software (MassLynx 4.1, Waters).

| Method validation
Validation of selectivity, linearity, sensitivity, accuracy, and precision was performed according to the European Medicines Agency (EMA) guidelines (EMA, 2012).

| Selectivity
Selectivity was assessed by analyzing serum samples from six different drug-free individuals without the addition of internal standard or analytes for evaluation of interference from endogenous components.
In addition, the drug-free serum sample was prepared with the internal standard working solution to evaluate purity and potential interferences affecting the analytes. Signal interference of less than 20% of the LLOQ and 5% of the internal standard was considered acceptable as per the EMA guidelines.

| Matrix effect, extraction recovery, and process efficiency
Two methods were used to evaluate matrix effects. Qualitative directly in water (set one) or added to serum samples from six drugfree individuals after (set two) or before (set three) the extraction procedure. The matrix effects were defined as the ratio between the mean peak areas of sets one and two, expressed as percentages.
Consequently, a calculated value of 100% indicates no measurable matrix effects. Recovery and process efficiency were determined in a similar way by the division of set three by set two and set three by set one, respectively. The calculated matrix effect should be within the interval 85-115%, and the extraction recovery should be more than 50% (Rudzki et al., 2018).

| Calibration model
Calibration curves were constructed by plotting the analyte/internal standard peak area ratio using the residual plot to compare linear and quadratic curve fit, with or without weighting of 1/x, for each compound resulting in a calibration curve with a coefficient of determination (r 2 ) > 0.995.

| Lower limit of quantification
Six replicates of the lowest calibration point 0.156 μmol/L followed by further dilution 1:1 with drug-free serum to 0.078 μmol/L (n = 6) were analyzed to determine the lower limit of quantification (LLOQ).

| Accuracy and precision
The accuracy and precision of the method were determined through repeated measurements of QC samples at three levels (n = 6). Intra-day and inter-day assay precision and accuracy were performed in one run and multiple runs, respectively. Accuracy was calculated as a bias, mean measured concentration divided by spiked concentration * 100 and precision as the coefficient of variation (CV%). Mean inaccuracy should be within 15% (20% for LLOQ) of nominal values for quality control samples. The imprecision should not exceed 15% (20% for LLOQ).

| Dilution integrity
To evaluate the dilution process performed on samples exceeding the highest calibrator, drug-free serum (n = 5) was spiked to 250 μmol/L and diluted fivefold with drug-free serum. According to EMA guidelines, accuracy should be within 85-115%, and precision should be within ± 15%.

| Carry-over
Carry-over was assessed by injecting 10 sample extracts of the lowest calibration standard, followed by a series of injections alternating between the highest and lowest calibrators (n = 10 per level). A t-test was used to compare the mean peak areas of the two sets of low calibration standards. Carry-over was considered insignificant if p > 0.05 (Honour, 2011).

| Stability
A system suitability check was used regularly before every batch confirming retention time, peak area, height, width, and the signal-tonoise ratio (S/N).
The stock solution and working solutions of antiviral drugs and internal standards' stability were evaluated after 6 months' storage at À80 C. The long-term storage stability of the patient samples over a time period of 6 months at À20 C was evaluated. Storage stability was claimed if the bias between the freshly prepared samples and samples stored for 6 months was ≤ 15%.

| Chromatography and sample preparation
Several C 18 , phenyl, and biphenyl stationary phases were tested in combination with methanol or acetonitrile with additives such as ammonium acetate, ammonium formate, acetic acid, or formic acid.
Our experiment showed that the biphenyl phase provided changes in selectivity relative to C 18 with a methanolic phase, but with acetonitrile-based phase the biphenyl behaved more similarly to a C 18 , which has been shown by Appulage et al. (Appulage et al., 2016). For these rather polar substances analyzed, in addition to interaction driven by the hydrophobic effect a biphenyl phase can give additional retention by π-πand cation-π interactions compared to a C 18 phase. A mobile phase consisting of 10 mmol/L ammonium acetate and methanol with a linear gradient on a biphenyl stationary phase gave the optimum separation and peak shape symmetry of

| Selectivity
Analyzing serum from six drug-free individuals showed an absence of endogenous interferences at the mass transitions at the retention times for ACV, CMMG, GCV, and PCV. Drug-free serum spiked with internal standards, ACV-IS, CMMG-IS, GCV-IS, and PCV-IS, showed no interfering peaks for ACV, CMMG, GCV, and PCV.

| Matrix effect, extraction recovery, and process efficiency
The qualitative matrix effect experiment showed no ionization suppression regions that would interfere with ACV, CMMG, GCV, or PCV. For CMMG, an ionization suppression region between 0.4 and 0.6 was found but with a retention time of 0.7 for CMMG and the use of CMMG-IS ensured a valid measurement of the analyte (Figure 2).
For the quantitative experiment the mean extraction recovery, matrix effect, and process efficiency are summarized in  Table 3. For uncompensated matrix effects, the CV ranged from 0.7 to 3.5% and for IS normalized matrix effects, the CV ranged from 0.5 to 3.3% for the six calibration levels.

| Accuracy and precision
Accuracy and precision of the analysis of QC samples at three concentration levels for each compound are presented in Table 4. The mean intra-day assay accuracy was 93 to 105% (n = 6), and inter-day assay accuracy ranged from 95 to 103% (n = 6) for ACV, CMMG, GCV, and PCV. CV of intra-day assay imprecision was 1.7-6.5%, and the interday assay imprecision was 1.4-4.2%.

| Dilution integrity
Drug-free serum was spiked with a concentration of 250 μmol/L for each of ACV, CMMG, GCV, and PCV and diluted to 62.5 μmol/L.
Accuracy and precision of the analysis were not affected as the fivefold diluted samples were measured with an inaccuracy of < 9.6% and imprecision < 7.1%.

| Carry-over
Application of the optimal washing procedure to prevent carry-over effects and ensure the precision and accuracy of the method was

| Stability
The analytes and internal standards were found to be stable for 24 h in autosampler at 5 C. Stock and working solutions of analytes and internal standards were stable for 6 months at À80 C. Patient samples stored at À20 C were found to be stable for 6 months, after being reanalyzed with freshly prepared calibration standards.
Unpaired two-sided t-test gave p-values of 0.827, 0.932, 0.999, and 0.929 for ACV, CMMG, GAN, and PNV, respectively, showing a good stability for up to 6 months' storage.

| Application of method
To evaluate method applicability, serum samples from patients undergoing treatment for herpes virus infection with ACV (n = 10) or GCV (n = 10) were analyzed.

| CONCLUSIONS
This work describes a fully developed and validated method for rapid quantification of ACV, its metabolite CMMG, GCV, and PCV, with the advantage of a short preprocessing, using a small amount of serum and protein precipitation with 1% formic acid in methanol containing corresponding isotopically labeled forms of the four substances analyzed as internal standards that result in high extraction recovery, T A B L E 4 Accuracy and precision for the quality controls for all included substances. From a single run on six replicates at each quality control level (inter-day assay) and from six different days (intra-day assay) Note: ACV, acyclovir; CMMG, 9-carboxymethoxymethylguanine; CV,coefficient of variation; GAN ganciclovir; Mean and SD in μmol/L; PCV, penciclovir; QC, quality control.
F I G U R E 3 Acyclovir (ACV) and 9-carboxymethoxymethylguanine (CMMG) concentrations during a 24-h sampling period collected from one subject given a single 200-mg tablet acyclovir minimized assay variation, and matrix effects. Further on, this study shows an improvement compared to previous methods by the use of a XEVO TQ-S micro for detection of the analytes with an LLOQ of 0.156 μmol/L for all four analytes. Optimal chromatographic performance, including selectivity and peak shape, was achieved using a column with a biphenyl stationary phase, resulting in a chromatographic runtime of 3 min. The novelty of using an alternative chromatographic selectivity by the choice of a core-shell biphenyl stationary phase demonstrated that this stationary phase could be an alternative even for these rather polar antiviral drugs.
In all, the validated LC-MS/MS method presented for the determination of the total concentration of ACV and its metabolite CMMG as well as GCV and PCV in human serum proved to be sensitive, specific, and accurate, making it suitable for therapeutic drug monitoring of these substances to improve patient treatment.

ACKNOWLEDGMENTS
The authors would like to express their great appreciation to Joline Ingelsson for valuable bioanalytical and technical support in the laboratory.