CA2425495A1 - Anti-retroviral analysis by mass spectrometry - Google Patents

Abstract

Methods for the simultaneous or sequential analysis and quantification of a plurality of antiretroviral analytes in a complex biological matrix by mass spectrometry are disclosed. The methods require minimal sample size, minimal preparation time and allow for rapid through-put.

Description

File N~. 31603-2031 TORYS LLP
Title: ANTI-RETROVIRAL ANALYSIS BY MASS SPECTROMETRY
Inventor: Steven J Soldin 6308 Waihonding Rd.
Bethesda, Maryland 20816, USA
t31041035:4tPM \lpicardup13t603~2029VeVOViralapplica6on13.doc r Title: ANTI-RETROVIRAL ANALYSIS 03Y MASS SPECTROnIIETRY
FIELD OF THE INVENTION
[0001] The present invention combines the fields of clinical medicine and analytical chemistry. In particular the irmention relates to the analysis of multiple analytes within a complex biological matrix by mass spectrometry, specifically where the analytes are antiretroviral drugs, including protease inhibitors (Pls), nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and fusion inhibitors (Fls).
DACKGROUND OF THE INVENTION

[0002] Quantification of the therapeutic level of drugs in the body is an important element of therapeutic drug monitoring (TDM), a process with numerous applications in clinical medicine. TDM has particular relevance in the treatment of Human Immunodeficiency Virus (HIV), which has become increasingly sophisticated and complex [1-3].

[0003] Over the past several years, there has been a rapid increase in the number of marketed anti-HIV drugs. Currently there are seventeen marketed antiretrovirals (Table 1 ), with several more expected to reach the market in the near future. Cocktails of 3 to 4 drugs are used routinely in anti-HIV therapy.
The specific choice of drugs depends on a number of factors, such as drug resistance, tolerability, drug interactions and effectiveness of treatment [4-6].
Combination of these factors (especially drug resistance) often leads to the need to alter the drugs prescribed to each patient. In such situations, therapeutic monitoring becomes an important factor.

[0004] The pharmacokinetics of many of the anti-HIV drugs are complex and sometimes unpredictable. Most of these drugs largely undergo oxidative .
CYP-3A4-mediated metabolism, which occurs primarily in the liver and gastrointestinal tract and is prone to numerous drug interactions and high inter and intra-individual variability.

[0005] Although some of the anti-HIV drugs do not require TDM
themselves, complicated regimens and drug cocktails with multiple drug interactions may justify TDM [7,8]. 9n addition, TDM may be the only way to effectively verify compliance, an issue which has been shown to be critical in HIV
therapy (9]. The literature has shown that TDM of antiretrovirals is very useful for the Pls and for the NNRTIs while its use for the NRTls may be predominately to assess patient compliance (10,11,12].

[0006] Traditionally, in clinical laboratories, TDM was performed by the use of immunoassay techniques and high performance liquid chromatography (HPLC).

[0007] However, immunoassay techniques are disadvantageous for the following reasons:
(1 ) Immunoassays are specific to each analyte. Therefore every drug must be analyzed separately.
(2) Numerous immunoassay kits must be purchased and procedures must be learned for each drug being analyzed by an immunoassay.
(3) Various instruments, such as gamma counters and photon counters, must be purchased to read the results from the immunoassays.
(4) The kits for immunoassays can be expensive.

(5) For some analytes, current immunoassays show, approximately, a 15 fold difference in results using kits from different manufacturers [13].
(6) In the case of a radioimmunoassay, precautions are necessary because of the radioisotopes involved.
(7) Immunoassays lack specificity and have problems with cross-reactivity, which tends to overestimate the concentration of the parent drug [13].

[0008] Recently, a method has been developed for the simultaneous determination of eight antiretroviral drugs (five Pls and three NNRTIs) by HPLC
ion trap mass spectrometry [15]. The authors of this method obtained calibration curves with a range of between nearly 20 and 10,000 ng ml-', and infra and interday precision of less than 15°l0. They also declared that their assay allows for elution and drug analysis within 15 minutes using 250 ~L of human plasma.

[0009] However, HPLC methods are disadvantageous for the following reasons:
(1 ) The method requires laborious and time consuming liquid-liquid extraction.
(2) The method lacks sensitivity and requires large serum volumes.
(3) Present HPLC methods are not applicable to NRTIs.
(4) Many different HPLC methods must be employed.

[0010] Researchers have applied liquid chromatography mass spectrometry methods to evaluate the plasma levels of various drugs and a number of assays for simultaneous quantification of various groups of anti-HIV

' _q._ drugs (especially Pls) have been reported [16-25]. However, none of these methods can assay Pls, NRTIs, NtRTIs, NNRTIs and Fls simultaneously.
[0011 Similarly, other liquid chromatography tandem mass spectrometry methods have been developed to measure the levels of up to five Pls in human plasma [23-24 ]. One of these methods involved precipitating plasma proteins and injecting the supernatant into a liquid chromai:ography column. The authors demonstrated a dynamic range of between 5.0 and 10,000 ng ml-', but used two calibration curves for each assay and 100 ~L of plasma was required.
SUMMARY OF THE INVENTION
[0012] The invention provides a fast, simple and accurate method for simultaneously or sequentially analyzing a plurality of antiretroviral drugs, including Pls, NRTIs, NtRTIs, NNRTIs and Fls, comprising ionizing the drugs and analyzing the drugs by mass spectrometry.
[0013] The drugs can be analysed in a variety of matrices, including plasma, serum, saliva and urine. For plasma and serum, both free and total drug analysis can be performed. Free form analysis can be performed for saliva, and for urine, the excreted amount can be measured.
[0014] Various ionization techniques are used in combination with analysis by mass spectrometry to arrive at this invention. Chromatographic separation is not required, although chromatography may be employed to clean the sample of impurities, such as salts.
[0015) There are several advantages to this invention:
(1 ) It permits the simultaneous or sequential quantification of a plurality of antiretroviral drugs, including Pls, NRTIs, NtRTIs, NNRTIs and Fls. Quantification of a range of antiretrovirals was beyond the _5_ reach of previous chromatography mass spectrometry methods, because of the high sensitivity and specificity demanded by the large variation of the various antiretroviral drug classes (See Table 2).
(2) The invention requires minimal sample preparation time. For example, after deactivating the HIV virus by a method known to one skilled in the art, preparing a sample of plasma for analysis can be completed within 20 minutes.
(3) The procedure does not require a large sample size. For example, a plasma sample a small as 80 gL permits quantitation of a plurality of HIV/AIDS drugs and allows for drug quantitation in infants and neonates.
(4) The procedure uses simple sample preparation techniques that are easy to use and highly reproducible, with good inter and intraday precision, below at least 7% for all ar~alytes.
{5) The invention permits the analysis of antiretrovirals in a sample of saliva or urine which permits simple sample acquisition and the remote submission of samples to a clinic for analysis. In other clinical methods, samples are taken by invasive means directly from the patient in a clinic X16).
(6) The time to complete an analysis of an batch of samples, from initiation to completion is about 15 minutes, which is far less time than alternative methods. Alternative methods take many hours.
(7) The invention is highly accurate over a wide range of concentrations, with calibration curves that are linear from at least 2 to 10,000 ng ml-' for stavudine, didanosine, zalcitabine and AZT;

100 to 10,000 ng ml-' for tenofovir; and from 10 to 10,000 ng ml's for all other antiretroviral drugs.
[0016] Accordingly, the invention provides a use for a mass spectrometer in simultaneously or sequentially analyzing a sample for a plurality of antiretroviral drugs in a fast, simple and accurate way. The sample may be, for example, serum, plasma, urine or saliva.
[0017] The invention provides a system for the fast, simple and accurate analysis of a plurality of antiretroviral drugs comprising: reagents for the preparation of the sample and reagents to peri~orm the analysis on a mass spectrometer, and the mass spectrometer to perform the analysis.
[0018] The invention also provides a kit, comprising the various reagents required for simultaneously or sequentially analyzing, within a sample, a plurality of antiretroviral drugs, including Pls, NRTIs, NtRTIs, NNRTIs and Fls. The kit may include a standard solution of the drugs of interest, compounds as internal standards, mobile phase solutions, HPLC column and quality control specimen.
For example, the kit can be used to test a sample for one or more of the seventeen commonly administered drugs listed in Table 1:
[0019] Accordingly, there is provided a method that allows for the simultaneous or sequential analysis of a plurality of antiretroviral drugs in a sample, comprising: obtaining a sample, deproteinating the sample, cleaning the sample, and analyzing and quantifying analytes within the sample by mass spectrometry.
[0020] Accordingly, there is also provided a method for the simultaneous or sequential analysis of a plurality of antiretroviral drugs, including Pls, NRTIs, NtRTIs, NNRTIs and Fls in a sample, including a sample of plasma, serum, saliva or urine, comprising: obtaining a sample, deproteinating the sample, _7_ cleaning the sample, and quantifying analytes within the sample by mass spectrometry.
[0021] Accordingly, there is also provided a method for the simultaneous or sequential analysis of a plurality of antiretroviral drugs, including Pls, NRTIs, NtRTIs, NNRTIs and Fls in a sample, including a sample of plasma, serum, saliva and urine, comprising: obtaining a sample, deproteinating the sample, cleaning the sample, and quantifying analytes within the sample by a mass spectrometry technique, absent chromatographic separation of the analytes.
[0022] Accordingly, there is also provided a use for a mass spectrometer in analyzing a sample for a plurality of antiretroviral drugs comprising:
obtaining a sample, deproteinating the sample, cleaning the sample and using the mass spectrometer to sequentially or simultaneously analyze the sample for antiretroviral drugs. The sample may be, for example, plasma, serum, urine or saliva.
DETAILED DESCRIPTION OF THE EXEMPLIFIED EMUODIMENTS
[0023] The invention provides methods for the simuUtaneous or sequentional analysis of a plurality of antiretroviral drugs, including Pls, NRTIs, NtRTIs, NNRTis and Fls, as well as variations, analogues and improvements thereto. A list of sample antiretrovirals that may be so analysed is provided in Table 1.
Sample preparation [0024) Any biological fluid can be used including plasma, serum, urine or saliva. A sample size of 80 ~L may be used.
[0025] The sample is de-proteinated, according to conventional techniques known to those skilled in the art. For example, a sample can be de-proteinated with acetonitrile, containing an internal standard as selected by one skilled in the art, followed by vortexing and centrifugation. Other methods of de-proteinization include precipitation with methanol, ethanol or salts.
Chromatography of sample [0026] The supernatant is introduced to a chromatography apparatus and eluted. In the case of a liquid chromatography apparatus, the column may be a C-18 column. No chromatographic separation is necessary for quantification where a tandem mass analyzer is employed. Where a tandem mass analyzer is employed, the matrix is cleaned of impurities by a chromatography column, or by other methods known to those skilled in the art. Optionally, a built in switching valve may be used.
fVlass spectrometery of sample [0027) The sample is then introduced into a mass spectrometer.
[0028] The following mass spectrometers can be used: any tandem-mass spectrometer, including the API 2000TM, the API 3000TM and the API 4000T"".
Instrumentation and ionization technigues (0029] The drugs are subjected to ionization. Various ionization techniques can be used. Preferably, atmospheric pressure chemical ionization is utilized, preferably using the heated nebulizer.
[0030) Ionization may be performed by utilizing the mass spectrometer in the negative or the positive mode, depending on a particular analyte's tendency to give rise to a particular ion form, as is known to those skilled in the art.
Optimally, the choice of negative or positive mode is made in accordance with Table 3. Optionally, cimetidine may be chosen as an internal standard, as it is suitable for both ionization modes, further simplifying the procedure.

_g_ (0031) As was discussed elsewhere [25], the use of the APCI source minimizes the potential for ionization suppression and matrix effects. Co-elution of the analytes and the internal standard further helps to overcome these problems, and eliminates the need to use internal standards structurally related to the analytes, which would be impossible considering the number of monitored drugs. Optionally, cimetidine may be chosen as an internal standard, as it is suitable for both ionization modes, further simplifying the procedure.
(0032] If the combination of drugs taken by the AIDS patient is not known, monitoring a plurality of antiretrovirals drugs (for example, monitoring seventeen or more) may be needed. This would result in a simultaneous monitoring at least fourteen ion transitions in the positive mode. !n such a case care should be taken to adjust dwell times and delays between ion transitions so that there are at least ten to twelve data points for each monitored peak. Failure to meet this requirement may result in inadequate peak integration and considerably worse precision. This, however, can occur only in the most complicated scenario, when the analyst does not know which anti-HIV drugs were taken by the patient, which is rarely the case in clinical practice.
Analysis [0033] Analytes, such as the antiretrovirals analysed by the present method, are identified in a mass spectrometer as is known to those skilled in the art. For example, analytes may be analyzed on the basis of the mass to charge ratio of their fragment ions. Preferably, an internal standard may be used, as is known to those skilled in the art. Calibration curves for known concentrations of drugs are established for comparison.
EXAMPLES
[0034] The invention may be demonstrated using the following three examples, provided to demonstrate but not limit the embodiments of the present invention. In the first example, 15 drugs are analyzed with the aid of a methanol standard. In the second example, 15 drugs (comprising the 15 analyzed in Example 1, and tanofovir, a NtRTI) are analyzed, with the aid of a serum standard. The third example illustrates how the invention might be applied to encompass new antiretroviral compounds, such as the FI T-20.
Example 9: l~lethanol Sfandard tVlatrix Standards and chemicals j0035] Standards of zalcitabine (ddC), didanosine (ddl) and zidovudine (AZT) were purchased from Sigma (St. Louis, M(~; USA). Primary standards of efavirenz, indinavir, nelfinavir, nevirapine, ritor~avir, saquinavir, lamivudine, abacavir and stavudine were obtained from the National Institutes of Heath (NIH) Aids Reagent Reference Program (McKessonHBaC BioServices, Rockville, MD) while standards for amprenavir, delavirdine and lopinavir were isolated from commercially available tabletslcapsules and characterized by I~-NMR, UV
spectroscopy and elemental analysis. Methanol, acetonitrile, and ammonium acetate were purchased from Sigma and were of HPLC grade.
Standard solutions and calibration curves [0036] Stock solutions were prepared separately to obtain concentrations of 0.1 mg mL-' for each drug (total o~f 15 drugs). Methanol was used as a solvent. Working Standard Solution was prepared by mixing equal amounts of stock solutions of each drug and diluting 1:4 with methanol to obtain a solution containing 1.67 pL g mL-' of each drug. A seven-point calibration curve (blank and six calibrators) was prepared for calibration. Along with 50 pL of the internal standard solution, 40 pL of working standard solutions were placed into 1.5 mL
Eppendorf conical plastic test tubes, after which 40 pL of blank plasma were added to each test tube. The calibrators were further treated as described under Sample Preparation.

[0037 A solution of 0.15 mg L~~ of cimetidine in methanol was used as an internal standard (for both negative and positive MS/MS modes).
Sample preparation [0038] For sample preparation, 80 mL of serum or heparinized plasma were placed into a 1.5 mL conical plastic Eppendorf test tubes containing 50 pL
of internal standard solution and vortexed briefly. Then 200 pL of acetonitrile were added, the test tubes were capped, vortexed vigorously for 30 seconds and cenrtifuged at 14,000 g for 10 minutes. The supernatant was transferred into autosampler vials for injection into the liquid chromatography tandem mass spectrometry system (LC/MS/MS). A doubleblank sample (a sample that contains neither of the standards nor the internal standard) was also prepared with each calibration curve. Sample preparation was performed at room temperature. Plasma obtained from HIV-infected patients was heated for 30 minutes at 56°C to deactivate the HIV virus [26].
[0039] This demonstrates a simple and expeditious method of preparing for analysis a complex matrix containing a plurality of antiretroviral drugs.
LC/MS/MS setup and procedure [0040] An API-2000TH" tandem mass spectrometer (SCIEX, Toronto, Canada) equipped with atmospheric pressure chemical ionization (APCI, heated nebulizer) source, two Perkin-Elmer PE-200TM series micropumps and autosampler (Perkin-Elmer, Norwalk, CT, USA) was used to perform the analysis. Data processing was performed on Analyst 1.1 software package (SCIEX). The main working parameters of the mass spectrometer are summarized in Table 4.
[0041] The procedure was based on an online extraction/cleaning of ,the injected sample with subsequent introduction into the mass spectrometer by using a built-in switching valve. 30 mL of the sample were injected onto a Supelco LC-18-DBTM (3.3 mm x 3.0 mm, 3.0 mL m ID) chromatographic column equipped with Supelco Discovery C-18T"" (3.0 mrn) guard column (Supelco, St.
Louis, MO, USA). The sample underwent cleaning with an aqueous solution of ammonium acetate (15 mM) at a rate of 1 mL mine'. After 2.4 min of cleaning the switching valve was activated, the column was flushed with methanal at a rate of 1 mL min-' and the sample was introduced into the mass spectrometer.
[0042] Analytes were then quantified by multiple reaction monitoring (MRM) (see Table 3 for MRM transitions). MRM allows for enhanced selectivity through the measurement of parent and daughter ions simultaneously for each of the compounds of interest.
[0043] Due to the high selectivity of the tandem mass analyzer, no chromatographic separation was necessary for quantification of the analytes and the analysis was complete less than a minute after activation of the switching valve. Total analysis time was 4.5 min, including equilibration time before the next injection. The procedure was completely automatic and controlled by the Analyst 1.1 software.
Drug interference studies [0044] The following commonly used drugs were tested at both their therapeutic and toxic concentrations for potential interference in the procedure described: acetaminophen, amikacin, caffeine, carbamazepine, digoxin, disopyramide, ethosuximide, flecainide, gentamicin, lidocaine, lithium, methotrexate, N-acetylprocainamide, phenobarbital, phenytoin, primidone, procainamide, quinidine, salicylate, theophylline, tobramycin, valproic acid, and vancomycin.

DrucLcomparison studies [0045, Comparison studies were performed for the P1s and NNRTIs using the tandem mass spectrometry method utilized all the BC Center for Excellence in HIV/AIDS.
Quality control and proficiency testina [0046] Serum or plasma spiked with known concentrations of the drugs and at three levels (low, medium and high) were used as daily quality controls.
External proficiency testing is available from "International Quality Control Program for Therapeutic Drug Monitoring in HIV Infection" (University Medical Center Nijmegan, Department of Clinical Pharmacy, PO Box 9101, 6500 HB
Nijmegan, The Netherlands).
[0047] Accuracy and precision were evaluated by analyzing quality control samples at low, medium and high concentrations on 20 different days. Within-run precision (%CV) was below 7% for all analytes. Between-day precision (%CV) was below 10% for all analytes at the tested concentrations. Accuracy (% of weighed-in target concentration measured) ranged between 95% and 105%. The results are summarized in Table 5. The assay was linear over the range of 2 to 2000 ng mL-' for stavudine, didanosine, zalcitabine and AZT, and 10 to 10000 ng mL-~ for all other drugs. None of the drugs listed above interfered in the method described.
[0048, The performance of the method was also compared with other analytical methods used for quantitation of nine of the tested drugs. A total of approximately 600 clinical samples containing various combinations of amprenavir, indinavir, saquinavir, delavirdine, efavirenz, lopinavir, ritonavir nelfinavir, and nevirapine were analyzed by the new method, as well as by a different LC/MS/MS method used routinely for the TDM of HIV drugs in British Columbia. The study was performed in a blinded manner and the laboratories did not share the results until after the completion of the study. Specimens were transported on dry ice and sent by overnight courier. They were stored at -70°C
until analyzed. These drugs are known to be stable at 4°C for '1 week.
The results are summarized in the Table 6. As can be seen from the Table 6, the results correlated very well for all of the tested drugs. Differences in the regression slopes can be explained by the differences in standardization between the two laboratories. Due to the difficulty i~ obtaining standards from the drug companies [16J, both of the laboratories purified the drugs initially from tablets. Gold standards for these drugs were only recently obtained by our laboratory from the NIH and were used to recalibrate our standards, perhaps accounting for some of the differences between the laboratories.
[0049) This demonstrates a simple and expeditious method of simultaneously analyzing a plurality of antiretroviral drugs by mass spectrometry.
' In particular, this demonstrates that the invention is easy and reliable and allows for the simultaneous measurement of any combination of AIDS drugs in less than minutes.
Example 2: Serum Standard Matrix [0050) Standards of zalcitabine (ddC), didanosine (ddl) and zidovudine (AZT) were purchased from Sigma (St. Louis, MO, USA). Primary standards of efavirenz, indinavir, nelfinavir, nevirapie, ritonavir, saquinavir, lamivudine, abacavir and stavudine were obtained from the N1H Aids Reagent Reference Program (McKessonHBOC BioServices, Rockville, MD) while standards for amprenavir, delavirdine, lopinavir and tenofovir were isolated from commercially available tabletslcapsules and characterized by H-NMR, UV spectroscopy and elemental analysis. Methanol, acetonitrile, and ammonium acetate were purchased from Sigma and were of HPLC grade.

[0051] Stock solutions were prepared separately to obtain a concentration of 1.0 mg mL'' of each drug (total of 16 drugs) with methanol as a solvent.
The working standard solution was prepared by mixing equal amounts of each drug and diluting with serum to obtain a solution containing 10,000 ng mL-' of each drug. The calibration curve consisted of a blank and five standards (10,000 ng ml-', 5,000 ng ml-', 2500 ng mL'', 1000 ng mL'', 100 ng mL''). Serum or plasma samples were obtained from hospital patients and proficiency testing samples (PT) from the Internationa) Quality Control Program for Therapeutic Drug Monitoring in HIV Infection (University Medical Center Nijmegan, Department of Clinical Pharmacy, PO Box 9191, 6500 HB Nijmegan, The Netherlands). Table 7 shows the concentration range covered by the six PT samples. A solution of 0.15 mg L'' of cimetidine dissolved in acetonitrile was used as the stock internal standard for both negative and positive MS/MS modes. The stock solution was diluted 2500 fold with acetonitrile to give the working internal standard solution.
[0052] For sample preparation, 80 pL of serum or heparinized plasma were placed into a 1.5 mL conical plastic Eppendorf test tubes containing 120 pL
of internal standard solution. The tubes were capped, vortexed vigorously for s and centrifuged at 14,000 g for 10 min. The supernatant was transferred into autosampler vials for injection into the LC/SIMS system. Sample preparation was performed at room temperature. Plasma obtained from I-iIV-infected patients was heated for 30 min at 56°C to deactivate the HIV virus [26J.
[0053] This demonstrates a simple and expeditious method of preparing for analysis a complex matrix containing a plurality of antiretroviral drugs.
Ionization and Soectrornet~
[0054] An API-2000T"" tandem mass spectrometer (SCIEX, Toronto, Canada) equipped with atmospheric pressure chemical ionization (APCI, heated nebulizer) source, two Perkin-Elmer PE-200TH series micropumps and autosampler (Perkin-Elmer, Norwalk, CT, USA) was cased to perform the analysis. An injection volume ofi 20 pL was used for all samples. Data processing was performed on Analyst 'i.1 software package (SCIE~). The main working, parameters of the mass spectrometer and detailed procedure have been described in Example 1, above.
[0055] Quantification of tenofovir by multiple reaction monitoring yielded an optimal transition in the positive ionization mode of 520.31270Ø See Table 3 fior the transitions for the other drugs.
[0056] Calibration curbes for all 16 anti-HIV drugs showed good linearity between a concentration range of 100 - 10,000 rig mL-~ (r>0.997 for all drugs).
The eight antiretrovirals present in the proficiency testing samples and their concentrations from low to high are listed in Table 7. Correlation of calibrators with the proficiency testing samples yielded the regression parameters listed in Table 8.
[0057] A method for obtaining extensive correlations for 9 of the 16 drugs with another method are described above in Example 1 and elsewhere [25]. At Children's National Medical Center in Vt/ashington, D.C., the therapeutic range for Pls is 150 ng mL-' - 6000 ng mL~', except for lopinavir which should be maintained at concentrations between 150 - 12,000 ng mL-' j27]. For the NNRTIs, the currently recommended therapeutic range is between 1200 and 7000 ng mL-~ [27].
[0058] This demonstrates a simple and expeditious method of simultaneously analyzing a plurality of antiretroviral drugs by mass spectrometry.
Example 3: Serum Standard Matrix [0059] The following example is provided to illustrate how the method can be applied to new antiretrovirals, such as Fls. Standard solution and sample preparation and analysis is performed. in accordance with the method set out in Example 2. A standard solution of a FI, for example T-20, is also prepared, with MRM transitions for T-20 established by a method known to one skilled in the art.
[0060] While the above detailed description describes the exemplifying embodiments of the present invention, it should be understood that the present invention is susceptible to modifications, variations and alterations without deviating from the scope of the invention.

Marketed anti-Hli/ drue~s Protease Nucleoside Non-Nucleoside Nuc6'eotide Fusion Inhibitors Reverse Reverse Reverse Inhibitors (PI) Transcriptase Transcriptase Transcriptase(FI) Inhibitors (NRTI)Inhibitors (NNRTI)(NtRTI) Amprenavir Abacavir Delavirdine Tanofovir T-20 Indinavir Didanosine (ddl~ Efavirenz Nelfinavir Lamivudine (3TC) Nevirapine f~itonavir Stavudine (d4T) Saquinavir Zalcitabine (ddC) Lopinavir Zidovudine (AZT) i Estimated limit of guantitation ~LOQ) versus known trough concentrations of anti-H11/ drugs Drug Estimated LOQ (ng/mL) Trough Concentration {nglmL) Efavirenz 1 -Zidovudine (AZT) 3 <20 Stavudine {d4T) 3 20 Indinavir <1 100 Abacavir 1 -Nelfinavir <1 1000 Delavirdine <1 3000-8000 Saquinavir <1 15--40 Nevirapine 1 3000 Lamivudine (3TC) 1 100-1000 Ritonavir <1 1000 Amprenavir <1 -Zalcitabine (ddC)1 Undetectable Didanosine (ddl) 1 100-300 Lopinavir <1 -MSIMS ion transitions and modes of analysis Drug Ionization mode MSIMS transition Efavirenz Negative 314.2/69.0 Zidovudine (AZT) Negative 2f6.21222.8 Stavudine (d4T) Negative 223.0142.0 indinavir Positive 614.5/421.3 Abacavir Positive 287.1 /191.1 Nelfinavir Positive 568.31330.1 Delavirdine Positive 457.21221.1 Saquinavir Positive 671.41570.3 Nevirapine Positive 267.21226.1 Lamivudine (3TC) Positive 230.11112.0 Ritonavir Positive 721.3/268.2 Amprenavir Positive 506.3/245.2 Zaicitabine (ddC) Positive 212.11112.0 Didanosine {ddf) Positive 237.1/137.1 Lopinavir Positive 629.31447.3 Tanofovir Positive 520.3/270.0 Tandem mass spectrometer main working parameters Parameter Value Nebulizer temperature, C 480 Dwell time per transition, 80 msec Nebulizer gas (Gas 10), psi 85 Auxiliary gas (Gas 2), psi 20 Curtain gas, psi 30 Nebulizer current, ~A 2 Ion energy, V 0.8 Inter-day accuracy and precision data n Mean(%) %
CV

nglmi nglming/ml ng/ml ng/mlng/ml Efavirenz 20 104.5 102.6101.4 9.9 8 6 Zidovudine 20 95.0 96.1 103 9 . .
(AZT) 2 5 . . 9.3 5.1 Stavudine (d4T)20 104 101 102 . . . 8.9 7.9 8.0 indinavir 20 104 98 101 . . . 7.6 6.0 3.2 Abacavir 20 96 97 97 . . . 8.7 6.2 5.5 Nelfinavir 20 95.9 104 102 . . 8.4 6.3 4.2 Delavirdine 20 104.3 96 99 . . 8.2 5.2 6.0 Saquinavir 20 103 101 98 . . . 9.5 7.1 4.1 Nevirapine 20 95.5 103 101 . . 6.9 4.2 5.9 Lamivudine 20 98.7 103 100 (3TC) 0 8 . . 8.8 4.9 2.1 Ritonavir 20 104 98 97 . . . 7.6 6.3 3.9 Amprenavir 20 103 99 99 . , . 8.6 7.4 4.5 Zalcitabine 20 96.4 101 103 (ddC) 9 0 . . 9.8 7.1 3.9 Didanosine 20 97.7 103 100 (ddl) 6 1 . . 9.7 6.1 6.5 Lopinavir 20 g5.0 103 102 . . 5.2 1.8 1.8 Linear regression parameters calculated from method comparisons n Slope Y-intercept X-interceptr Sy,X

Amprenavir16 0.54 0.06 270.8 146.1 -498.7 0.911 304.0 Delavirdine10 0.79 0.03 266.6 231.4 -338.1 0.993 425.9 Nevirapine28 0.78 ~- 506.5 230.3 -546.0 0.928 428.5 0.07 Lopinavir 32 0.87 0.05 558.0 381.1 -642.9 0.949 944.2 Efavirenz 15 1.15 0.08 150.8 188.8 -131.5 0.971 398.4 Ritonavir 52 0.96 ~- -4.8 44.4 -5.05 0.992 266.7 0.02 Saquinavir26 0.91 0.03 11.0 18.4 -12.0 0.989 71.08 Nelfinavir33 0.73 0.02 88.5 41.5 -121.7 0.988 146.8 Indinavir 98 1.16 0.02 -77.1 34.4 -66.6 0.983 252.2 Sy,X- standard deviation of the residuals.

Linear regression aarameters calculated from comparison of our results with the testing samples or 6 known target concentrations in proficiency Drug n Siope 1'-intercept r Sy,X

Amprenavir6 0.93 0.02 -14.0 105.6 0.998180.5 Indinavir 5 0.92 0.06 -30.3 43~i.1 0.993699.7 Lopinavir 6 1.03 0.05 268.6 341.9 0.995490.0 Nelfinavir6 1.07 -~ 0.02 -161.9 4~-.481.00077.06 Ritonavir 6 0.94 0.03 -77.5 205.2 0.998363.0 Saquinavir5 0.95 0.03 -50.93 91.29 0.998141.4 Nevirapine5 0.94 0.19 -67.3.1 727.90.944958.4 Efavirenz 5 1.09 0.02 138.7 109.2 0.999144.4 Target concentrations y testing ) les of proficienc (PT samp ~rug Concentration Levei mL-') (ng Low Medium High PT target values(1 (2) (3) (4) (5) (6) ) Amprenavir 220 240 2150 2160 86007200 Indinavir 170 130 2330 1010011720 Lopinavir 1000 1170 5000 470 1000011680 Nelfinavir 230 320 1640 1530 21206360 Ritonavir 270 240 2670 2420 13400 Saquinavir 120 110 1410 34005080 Nevirapine 500 1090 3900 3160 7800 Efavirenz 460 1000 3700 73006590 CITED REFERENCES
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Claims (32)

1. A method for mass spectrometric analysis of a sample comprising at least two antiretroviral drugs from at feast two classes of antiretroviral drugs, the method comprising the steps:
(a) providing a sample comprising at least two antiretroviral drugs from at least two classes of antiretroviral drugs;
(b) introducing the sample comprising at least two antiretroviral drugs from at least two classes of antiretroviral drugs into a mass spectrometer; and (c) analyzing the sample using the mass spectrometer.

2. The method according to claim 1 wherein the mass spectrometer is a tandem-mass spectrometer.

3. The method of claims 1 or 2 further comprising a step of deproteinating the sample.

4. The method according to any of claims 1 to 3 wherein the classes of antiretroviral drugs are selected from the group consisting of Pls, NRTIs, NtRTIs, NNRTIs and Fls.

5. The method according to any of claims 1 to 4 wherein the antiretroviral drugs are selected from the group consisting of amprenavir, indinavir, nelfinavir, ritanavir, saquinavir, lopinavir, abacavir, didanosine, lamivudine, stavudine, zalcitabine, zidovudine, delavirdine, efavirenz, nevirapine, tanofavir and T-20.

6. The method according to any of claims 1 to 5 wherein the sample containing at least two antiretroviral drugs is obtained from a biological matrix selected from the group consisting of plasma, serum, urine and saliva.

7. The method of ciaim 6 wherein the biological matrix is plasma.

8. The method of claim 6 wherein the biological matrix is serum.

9. The method of claim 6 wherein the biological matrix is saliva.

10. The method according to any of claims 1 to 9 wherein size of the sample containing at least two antiretroviral drugs is about 80 µL.

11. The method according to claim 3 wherein the step of deproteinating the sample comprises:
(a) adding acetonitrile to the sample;
(b) vortexing the sample; and (c) subjecting the sample to centrifugation.

12. The method according to claim 3 wherein the step of deproteinating the sample comprises subjecting the sample to precipitation with an agent selected from the group consisting of methanol, ethanol and salt.

13. The method of any of claims 1 to 12 further comprising a step of cleaning the sample.

14. The method according to claim 13 wherein the step of cleaning the sample comprises introducing the sample to a chromatography apparatus and eluting the sample.

15. The method according to claim 2 wherein the tandem-mass spectrometer is selected from the group consisting of API 2000.TM., API 3000.TM. and API
4000.TM..

16. The method according to claim 1 wherein the step of analyzing the sample using a mass spectrometer comprises a step of atmospheric pressure chemical ionization using a heated nebulizer.

17. The method according to claim 1 wherein the step of analyzing the sample using a mass spectrometer comprises multiple reaction monitoring.

18. The method according to any of claims 1 to 17 which does not include chromatographic separation of the antiretroviral drugs.

19. The method according to any of claims 1 to 18 wherein the sample comprises a plurality of antiretroviral drugs and they are analyzed simultaneously.

20. The method according to any of claims 1 to 19 wherein the sample comprises a plurality of antiretroviral drugs and they are analyzed sequentially.

21. A method for therapeutic drug monitoring in patients with HIV infection, comprising:
(a) providing a sample comprising at least two antiretroviral drugs from at least two classes of antiretroviral drugs;
(b) introducing the sample comprising at least two antiretroviral drugs from at least two classes of antiretroviral drugs into a mass spectrometer; and (c) analyzing the sample using the mass spectrometer.

22. The method according to claim 21 wherein the mass spectrometer is a tandem-mass spectrometer.

23. The method of any of claims 21 and 22 further comprising a step of deproteinating the sample.

24. The method according to any of claims 21 to 23 wherein the classes of antiretroviral drug is selected from the group consisting of Pls, NRTIs, NtRTIs, NNRTIs and Fls.

25. The method according to any of claims 21 to 24 wherein the antiretroviral drug is selected from the group consisting of amprenavir, indinavir, nelfinavir, ritonavir, saquinavir, lopinavir, abacavir, didanosine, lamivudine, stavudine, zalcitabine, zidovudine, delavirdine, efavirenz, nevirapine, tanofavir and T-20.

26. The method according to any of claims 21 to 25 wherein the sample containing at least two antiretroviral drugs is obtained from a biological matrix selected from the group consisting of plasma, serum, urine and saliva.

27. The method according to any of claims 21 to 26 further comprising a step of cleaning the sample.

28. A system for the mass spectrometric analysis of a sample comprising at least two antiretroviral drugs from at least two classes of antiretroviral drugs, comprising:
(a) reagents for deproteinating the sample;
(b) reagents far analyzing the sample by mass spectrometry; and (c) a mass spectrometer.

29. A kit for use in mass spectrometric analysis of a sample comprising at least two antiretroviral drugs from at least two classes of antiretroviral drugs, comprising:
(a) reagents for deproteinating the sample;
(b) reagents for analyzing the sample by mass spectrometry;
(c) instructions for analyzing the sample using a mass spectrometer.

30. The kit according to claim 29 further comprising:
(a) mobile phase solutions;
(b) a chromatography column; and (c) a quality control specimen.

31. Use of a mass spectrometer for sequentially or simultaneously analyzing a sample containing at least two antiretroviral drugs from at least two classes of antiretroviral drugs.

32. The use of claim 31 wherein the mass spectrometer is a tandem mass spectrometer.