FIELD OF THE INVENTION
The present invention relates to a high performance liquid chromatography tandem mass spectrometric method for the determination of Atovaquone in human plasma using protein-precipitation technique.
BACKGROUND OF THE INVENTION
Atovaquone. 2-[4-(4-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone is a widely used antiprotozoal and is potently active (in animals and in vitro) against Pneumocystis carinii, Plasmodia, and tachyzoite and cyst forms of Toxoplasma gondii Atovaquone, a highly lipophilic compound resembling ubiquinone, has a low aqueous solubility, and that is the reason for the poor bioavailability of Atovaquone after oral administration. It is reported that after a single oral dose, absorption of the drug is slow and erratic; it is increased about three-fold by the presence of fatty food and is dose-limited above 750 mg.
US 4.981,874 discloses the use of Atovaquone against Pneumocystis carinii infection in a mammal. EP 123,238 and US 5,053,432 disclose the use of Atovaquone against Plasmodium falciparum and also against Eimeria species such as E. tenella and E.acervulina which are causative organisms of coccidiosis. Further, the use of Atovaquone against Toxoplasmosis and Cryptosporidiosis is disclosed in EP 445,141 and 496,729 respectively.
Several attempts have been made in prior art to develop a sensitive, selective and high throughput bioanalytical method for the measurement of Atovaquone in human plasma. However, commercially available methods were found tedious, lacking sensitivity and require a lengthy run time.
An article published in The Ann. Pharmacother. Vol. 32 (1998), 1004, by Freeman et al discloses a HPLC method for the determination of Atovaquone in plasma where low level quantification limit of 500ng/mL for 750mg/5mL of suspension was observed as well as a lengthy analysis run time exceeding more than 8 minutes was noted.
An article published in Journal of Chromatography Vol. 652 (1994), 211-219 by DeAngelis et al disclose a liquid-liquid extraction method for determination of Atovaquone in plasma. This method was found to be a time consuming method and required handling of huge organic solvents.
Another article published in Journal of Chromatography Vol. 678 (1996), 297-302 by Hannan et al discloses the determination of Atovaquone in plasma by reverse-phase high-performance liquid chromatography using liquid-liquid extraction The sensitivity achieved in this method was 100ng/mL, however, validation data presented was inadequate and run time analysis was more than 6 minutes.

Another article published in Journal of Chromatography Vol. 675 (1996), 180-182, by Hansson et al involving a protein precipitation-HPLC assay with high injection load of 20ul showed low precision because of column degradation due to incomplete removal of protein in the precipitation step.
SUMMARY OF THE INVENTION
To overcome all these problems, we have now developed a rapid sensitive, high performance liquid chromatography tandem mass spectrometric method for the determination of Atovaquone in human plasma using protein precipitation technique. This new method has high sensitivity and specificity, requires very short analytical run time and simultaneously achieves excellent extraction efficiency as compared to other methods known in the prior art for the measurement of Atovaquone in human plasma, involving minimum plasma volume for sample processing leading to minimum blood collection from volunteers.
According to one of the aspect, there is provided a high performance liquid chromatography tandem mass spectrometric method for the determination of Atovaquone in human plasma using protein-precipitation technique.
In another aspect, there is provided a high performance liquid chromatography tandem mass spectrometric method for the determination of Atovaquone in human plasma using protein precipitation technique wherein Lapachol is used as an internal standard and mass spectrometer uses heated nebulizer ion source in negative ion mode.
According to one of the aspect, there is provided a high performance liquid chromatography tandem mass spectrometric method for the determination of Atovaquone comprising the steps of:
a. Mixing Atovaquone plasma samples with a known quantity of Lapachol solution and vortexing the plasma samples to obtain the clear supernatant;
b Injecting the clear supernatant formed in step a) into HPLC autoinjector, wherein the mobile phase consists of a mixture of formic acid and acetonitrile and mass spectrometer uses heated nebulizer ion source in negative ion mode.
DETAILED DESCRIPTION
Atovaquone" as employed herein is intended to include isomers, cis and trans forms of Atovaquone or mixture thereof or any pharmaceutically acceptable salts thereof. Atovaquone may be used in any of the polymorphic forms such as Form I or III It may be used alone or in combination with Proguanil.
As used herein, the term "pharmaceutically acceptable salts" refers to inorganic base salts such as alkali metal (e.g. sodium and potassium) salts and alkaline earth metal (e.g. calcium) salts; organic base salts e.g. phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine and diethanolamine salts; and amino acid salts e.g. lysine and arginine.
High performance liquid chromatography tandem mass spectrometric (LC/MS/MS) is an analytical technique generally used in pharmaceutical analysis. In this technique, solutions derived from samples are injected onto an HPLC column, compounds are separated on the basis of their relative interaction with the stationary phase and the mobile phase, components eluting from the chromatographic column are then introduced to the mass spectrometer via a specialized interface. The two most common interfaces used for HPLC/MS are the electrospray ionization and the atmospheric pressure chemical ionisation interfaces (APCI). In the present invention, APCI mode was used as it has primary applications in the areas of ionisation of low mass compounds such as Atovaquone. A triple-quadrupole mass spectrometer (MS-MS) was used with APCI source and channel electron multiplier (CEM) detector in negative ion detection mode. In APCI source, the temperature of the heater in the source was maintained at 500 C with nebulizer current at -2.00 and nebulizer gas at 10 psi (zero air) for the better vaporization of HPLC eluent.
Protein precipitation is a one-step extraction method for the removal of protein from the drug containing plasma samples prior to LC/MS/MS. In our case drug is highly protein bound -99 9%, hence protein precipitation technique is the most suitable pathway for Atovaquone extraction. Protein precipitation causes Atovaquone to unbound from protein and be present in the clear supernatant. Acetonitrile and formic acid causes the protein precipitation to occur during the sample preparation stage
Lapachol is used as an internal standard due to structural similarity between Atovaquone and Lapachol, both belong to chemical class of naphthoquinone. Lapachol internal standard is prepared by dissolving accurate amount of Lapachol in methanol to make 1mg/mL, which is further diluted with formic acid and acetonitrile mixture.
Mobile phase used for HPLC is a mixture of 0.1% formic acid solution (v/v) and acetonitrile in the ratio of 20:80. It was found that the addition of formic acid increased signal without affecting noise levels
The LC/MS/MS method for the determination of Atovaquone was validated. The method demonstrated excellent high sensitivity of 50.3 ng/mL as the lower limit of detection (LLOQ) and dynamic linearity ranging from 50.3 ng/mL to 23924.6 ng/mL with acceptable precision and accuracy, with a run time of around 2.5 min
In order to further illustrate the present invention, a detailed prototype analysis method is provided below However, the prototype is for the purpose of illustration and should not be construed as limiting the scope of the present invention.
EXAMPLES Example 1
I.Preparation of Solutions
1 Preparation of Atovaquone Standard Stock solution
Atovaquone working standard was accurately weighed and dissolved in 50% of N, N-Dimethyl formamide in methanol to make a solution of approximately 3 mg/mL
2 Preparation of Lapachol Stock solution
Lapachol working standard was accurately weighed and dissolved in methanol to make a solution of approximately 1 mg/mL. Stock dilution of Lapachol of appropriate concentration was prepared using Solution-2 as diluent (described below).
3 Preparation of Solution-1 (0.1% formic acid solution): 1 mL of formic acid was transferred into 1000 mL volumetric flask and volume was made up with HPLC Grade water to get 0.1% formic acid solution. The solution was mixed well and ultrasonicated.
4 Preparation of Solution-2 (0.2% formic acid solution): 2 mL of formic acid was transferred into 1000 mL volumetric flask and the volume was made up with acetonitrile.
5 Preparation of Mobile Phase
Mobile Phase: 200 mL of solution-1 was transferred into a reagent bottle and 800mL of acetonitrile was added. It was then well mixed, ultrasonicated and degassed.
6 Preparation of calibration standards
Calibration standards were prepared by spiking appropriate analyte stock solution in blank citrate.phosphate,dextrose,adenine (CPDA) plasma.
7. Preparation of samples
Plasma samples that were stored at -50°C were retrieved and allowed to thaw at room temperature before processing After thawing, 100uL of vortexed plasma samples were aliquoted in a 1.7 mL eppendorf tube, followed by addition of 1000uL of a 250.0 ng/mL solution of Lapachol and mixed thoroughly. Samples centrifuged at 14,000 rpm for 5 minutes with temperature between 4 C to 10 C. The clear supernatant of 500 01/8µL was transferred to an autosampler vial and 10 µL was injected into the LC/MS/MS system.
8 Chromatographic conditions
LC separation was performed on a Synergi 4µ Polar-RP 80A (150 x 2.0mm, 4µ) column (Phenomenex, Torrance, CA, USA). The mobile phase consisting of a mixture of 0.1 % formic acid solution (v / v) and acetonitrile (20: 80 v / v) was delivered at a flow rate of 0.5mL / min. The column was thermostatically controlled to a temperature of 45°C ± 1.0°C and sample cooler temperature
was maintained at 10°C ± 1.0°C. The injection volume was 10uL Retention time for Atovaquone: 1.0 to 2 minutes and Lapachol: 0.6 to 1.6 minutes was set as validation parameter.
9 Mass spectrometric conditions
A triple-quadrupole mass spectrometer (MS-MS) was used with APCI source and channel electron multiplier (CEM) detector in negative ion detection mode. In APCI source, the temperature of the heater in the source was maintained at 500 C with nebulizer current at -2.00 and nebulizer gas at 10 psi (zero air) for the better vaporization of HPLC eluent. For the transition of ions from atmosphere to vacuum region, curtain gas (ultra high purity nitrogen) was served at 8 psi for the effect of collisional induced dissociation (CID) in the curtain plate region. For the precursor ion fragmentation, collisional gas (ultra high purity nitrogen) was served at 5 psi for the effect of collisional activated dissociation (CAD) in the collision cell. MRM mode was used for scanning throughout this study. The transitions selected were m I z 365.2^ m I z 337.1 and m I z 240.9^ mlz 185.7 for Atovaquone and Lapachol respectively, with a dwell time of 200 ms per transition.
The method was validated in terms of selectivity, linearity, sensitivity, precision, accuracy, recovery and stability and was found to be acceptable according to guidelines issued by FDA for validation of bioanalytical methods.
Three precision-accuracy batches were run to check intra and inter- day precision and accuracy. Each batch of spiked plasma samples included one complete calibration curve (consisting of two blank plasma, two blank plasma with internal standard and eight different non-zero concentration standards) and six replicate quality control samples at LOQQC, low, medium and high concentrations (Table 1) The percentage recovery of Atovaquone was evaluated by measuring the peak area response of extracted quality control samples at low, middle and high concentrations against the peak area response of unextracted quality control samples of equivalent concentrations. The recovery of Atovaquone was found to be more than 80%.
Table 1: Precision and accuracy of the method for determining Atovaquone concentration in plasma samples (SD-deviation)
(Table Removed)

WE CLAIM:
1. A high performance liquid chromatography tandem mass spectrometric method for the determination of Atovaquone in human plasma using protein-precipitation technique.
2 The high performance liquid chromatography tandem mass spectrometric method according to claim 1 wherein protein precipitation is caused by Acetonitrile and formic acid.
3 The high performance liquid chromatography tandem mass spectrometric method according to claim 1 wherein Lapachol is used as an internal standard.
4 The high performance liquid chromatography tandem mass spectrometric method according to claim 1 wherein ionization in mass spectrometer is produced by atmospheric pressure chemical ionization.
5. The high performance liquid chromatography tandem mass spectrometric method according to claim 1 wherein the method has analysis run time of less than 2.5 minutes.
6 The high performance liquid chromatography tandem mass spectrometric method according to claim 1 wherein a mixture of acetonitrile and formic acid is used as mobile phase in the high performance liquid chromatography.
7 The high performance liquid chromatography tandem mass spectrometric method according to claim 1 wherein the method has a sensitivity of about 50 ng/mL.
8 The high performance liquid chromatography tandem mass spectrometric method according to claim 1 wherein the mass spectrometer has negative ion heated nebulizer.
9 The high performance liquid chromatography tandem mass spectrometric method according to any of the preceding claims wherein the method comprises the steps of:

a) Mixing Atovaquone plasma samples with a known quantity of Lapachol solution and vortexing the plasma samples to obtain the clear supernatant; and
b) Injecting the clear supernatant formed in step a) into HPLC autoinjector.
10 The high performance liquid chromatography tandem mass spectrometric method for the
determination of Atovaquone in human plasma as used and exemplified herein.