The column is crucial

Ganciclovir was developed initially as a drug for the treatment of cytomegalovirus infection, after which it was discovered that GAN could inhibit in vitro other herpes viruses (human herpes viruses 1 and 2 types, Epstein-Barr viruses, chickenpox virus, human herpes virus type 6 etc). [1] Typically, ganciclovir was used intravenously only during a therapy, and a new prodrug named valganciclovir was developed to increase the bioavailability. Valganciclovir metabolizes easily into ganciclovir which in turn provides a therapeutic effect. [2, 3]

Like many other antiviral drugs, valganciclovir and ganciclovir are both rather hydrophilic substances, as clearly shown by the values of their octanol/water partition coefficients (log P, table 1). These properties must be considered in the development of analysis methods including sample preparation and separation techniques.

Previous analytical methods using HPLC with UV detection [7, 8, 9] and HPLC coupled with mass spectrometer [10] were not able to measure valganciclovir and ganciclovir simultaneously, rather only one substance at a time.

Optimal chromatographic separation with MS detection

The authors of this publication tried to develop a method for simultaneous determination of GAN and VAL using HPLC-UV, but this method required the use of a specific “YMC-Pack Polyamine II” chromatographic column and setting of eluent flow at 2 mL/min, which significantly increased reagent consumption. Moreover, analysis time was quite long (about 26 minutes) to achieve optimal separation of the substances and internal standard.

The combination of MS detection and chromatographic separation enables reducing the flow rate and significantly reducing the analysis time. This study provides development and validation of the methods for the determination of valganciclovir and ganciclovir in human blood plasma by LC-MS/MS.

Materials and methods

The UHPLC Nexera XR coupled with tandem mass spectrometer LCMS-8040 (both from Shimadzu) were used. Methanol (UHPLC-grade) was purchased from J.T.Backer, acetonitrile (LCMS-grade) from Biosolve, formic acid (98 % pure) and aqueous ammonia (“for analysis” grade) from PanReac. Deionized water was produced by “Milli-Q” system from Millipore. Valganciclovir hydrochloride (USP reference standard, 99.2 %), ganciclovir (USP reference standard, 97.5 %) and acyclovir (ACI) applied as an internal standard (USP reference standard, 94.6 %) were used to prepare stock solutions by dissolving in methanol. Mixed working solutions of GAN and VAL and an ACI working solution were prepared by diluting of stock solutions with the same solvent to the required plasma concentrations (table 2). Stock and working solutions as well as intact blood plasma samples were stored prior to use in a freezer at -45 ^oC.

Sample preparation

10 μL of acyclovir working solution was added to 200 μL of a calibration sample placed in 2 mL Eppendorf centrifuge tubes, 600 μL of acetonitrile were then added, vortexed for ten seconds and centrifuged for 15 min at 13,500 rpm. The resulting supernatant was transferred into vials and placed in the autosampler.

HPLC conditions

Column:	Shim-Pack GWS C18 (5 µm; 150 x 4.6 mm)
Column temperature:	40 °C
Mobile phase (A):	0.1 % formic acid in water with 0.08 % ammonia (v/v)
Mobile phase (B):	0.1 % formic acid, 10 % water in acetonitrile with 0.08 % ammonia (v/v)
Gradient:	The gradient is shown in table 3
Injection volume:	5 μl

MS conditions

Ionization:	ESI, positive
Nebulizing gas:	3 L/min
Drying gas:	20 L/min
Heater block:	400 oC
Desolvation line:	200 oC
VAL:	355.20 m/z → 152.05 m/z
GAL:	255.80 m/z → 152.00 m/z; 255.80 m/z → 134.90 m/z
ACI:	226.00 m/z → 152.00 m/z; 226.00 m/z → 134.95 m/z
Registration time:	0.00 – 7.00 min

Results and discussion

Method development

Development of a chromatographic separation method for valganciclovir and ganciclovir is challenging due to their weak retention on “classic” octadecyl columns. Use of the columns with amino groups also did not provide good retention and proper separation of VAL or GAL. An idea was to test C18 columns with different total carbon content (table 4). The best results were achieved using Shim-Pack GWS column with the lowest total carbon content. Despite the insignificant retention of valganciclovir and ganciclovir on this column, the chromatographic peaks were separated from the dead volume, and the method met the requirements of the regulation for the validation, including the “selectivity” parameter.

Method validation

The method was validated based on the guidelines for the examination of medicines [12] as well as FDA [13] and EMA [14] guidelines for the following parameters: selectivity, matrix effect, calibration curve, accuracy, precision, recovery, lower limit of quantification, carry-over and stability.

Selectivity

Six samples of intact plasma obtained from different sources were tested as well as samples of intact plasma spiked with working solutions of valganciclovir and ganciclovir (5 and 50 ng/mL respectively). Additionally, samples of intact plasma with hemolysis and samples with increased lipid content were analyzed. With blank plasma chromatograms (figure 3) intensity of the peaks with the same retention time as the target substances did not exceed 20 % of the signal at LLOQ (Lower Limit Of Quantification) and 5 % of the internal standard signal.

Matrix effect

The samples including working solutions of valganciclovir, ganciclovir and acyclovir as well as spiked plasma samples were examined to estimate a matrix effect. It was estimated at low (L) and high (H) concentration levels (see table 2) for both valganciclovir and ganciclovir and at concentration of 1,000 ng/mL for acyclovir. The results are shown in tables 5 and 6.