CHARACTERIZATION OF PROSTAGLANDINS
INTRODUCTION TO THE INVENTION
The present invention relates to characterization of prostaglandins. More particularly, the present invention relates to use of a non-steroidal anti-inflammatory drug ("NSAID") during determination of prostaglandin content in a biological fluid.
Prostaglandins ("PG") are 20-carbon fatty acids that are produced in a variety of tissues and that mediate an array of physiologic and pathologic processes. Prostaglandins are broadly classified as PGA PGB, PGC, PGD, PGE, PGF, PGG, and PGH based on their cyclopentane/pentene ring substitution patterns. Each general PG class is sub-classified based on the degree of unsaturation (e.g., PGEi, PGE2, PGF2).
Prostaglandins are important mediators of normal physiologic events and have been implicated in a variety of pathologies like inflammation, pain, pyrexia, cardiovascular disease, renal disease, cancer, glaucoma, allergic rhinitis, asthma, preterm labor, male sexual dysfunction and osteoporosis. This has led to the development of a number of PG drug compounds including gemeprost, beroprost, misoprostol, enoprostil, omoprostil, limaprost, litanoprost, travoprost, and the like.
Misoprostol (Formula I) has a chemical name (11a,13E)-11,16-dihydroxy-16-methyl-9-oxoprost-13-en-1-oic acid methyl ester (synthetic derivative of PGEi). Another chemical name for the compound is (±)-methyl-11a,16-dihydroxy-16-methyl-9-oxoprost-13E-en-1-oate. The compound is a viscous oily liquid having cytoprotective action. It is used in the treatment of benign gastric and duodenal ulceration and in the prevention of NSAID-induced ulcers.

Misoprostol is commercially available in CYTOTEC® (G.D. Searle & Co., USA) products, which are an oral tablets containing 100 ^g or 200 i^g of the compound. A combination product of diclofenac sodium and misoprostol is commercially available under the brand name ARTHROTEC® (G.D. Searle & Co., USA).

Orally administered misoprostol is rapidly absorbed and instantly metabolized to its active metabolite, misoprostol acid. Misoprostol acid content in blood is determined to evaluate pharmacokinetic parameters.
Determination of misoprostol acid content in various biological fluids is a difficult task owing to the very low therapeutic dose of misoprostol. Various analytical techniques have been reported for the determination of misoprostol and its metabolite (misoprostol acid) content in biological fluids (including serum, breast milk and blood). For example, see Bernhard Watzeret al., "Determination of misoprostol free acid in human breast milk and serum by gas chromatography/negative ion chemical ionization tandem mass spectroscopy ("GCMS")," J. Mass Spectroscopy, 2002, 37(9), 927-933; K. Gemzell Danielsson et al., "Comparison between oral and vaginal administration of misoprostol on uterine contractility," Obstetrics & Gynecology, 1999, 93(2), 275-280; and Y. Zou et al., "Determination of misoprostol acid in human plasma by liquid chromatography coupled to tandem mass spectrometry," Journal of Chromatography B, 2007, 852(1) 122-127.
For an analytical method to be acceptable, regulatory agencies require that interferences during the determination of an analyte must be significantly low. United States Food and Drug Administration ("FDA") Center for Drug Evaluation and Research ("CDER") guidance to industry titled 'Bioanalytical Method Validation' (published May 2001) recommends that the interferences during the determination of an analyte in a biological sample should be less than the lower limit of quantitation ("LLOQ") for a particular analytical method employed.
LLOQ is the lowest concentration of the standard curve that can be measured with acceptable accuracy and precision. The lowest standard on the calibration curve should be accepted as the limit of quantification if the following conditions are met:
a) The analyte response at the LLOQ should be at least 5 times the response compared to blank response.
b) Analyte peak (response) should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80-120%.
Accordingly, the present invention fills a gap in available technologies by providing an analytical method with minimized interference during determination of misoprostol and misoprostol acid content in biological fluid.
This and other such needs are addressed by the instant invention.

SUMMARY OF THE INVENTION
The present invention relates to use of an NSAID during determination of prostaglandins content in a biological fluid.
An aspect of the present invention provides for use of an NSAID for minimizing the analytical interference during determination of prostaglandin content in a biological fluid.
Another aspect of the present invention provides for use of an NSAID in the sample preparation for minimizing the analytical interference during determination of misoprostol acid content in a biological fluid.
A still further aspect of the present invention provides for use of an NSAID during sample preparation for minimizing the interference to an extent less than about the lower limit of quantitation of analytical method during determination of misoprostol acid content in a biological fluid.
In an embodiment, the present invention provides a method for determining the prostaglandin content in blood with minimized analytical interference, wherein said method comprises:
a) adding an NSAID to a sample of blood and separating plasma from the blood; and
b) determining the prostaglandin content in plasma by a liquid chromatographic ("LC") technique.
In another embodiment, the present invention provides a method for determining a misoprostol acid content in blood with minimized analytical interference, wherein said method comprises:
a) adding an NSAID to a sample of blood and separating plasma from the blood, or separating plasma from blood and adding an NSAID; and
b) determining the misoprostol acid content in plasma by a high performance liquid chromatography tandem mass spectrometry("HPLC LC-MS/MS") technique.
In still further embodiment of the present invention, a HPLC LC-MS/MS technique for determining a misoprostol acid content in plasma comprises:
a) extracting misoprostol acid from plasma and dissolving it in a reconstitution solution comprising acetonitrile and a 0.2 % aqueous formic acid solution (80:20 v/v ratio);

b) injecting the sample solution onto a reversed phase ("RP") C18, 3.5 jjm HPLC column,
c) eluting misoprostol acid from the column using a mobile phase comprising acetonitnle and a 0.2 % aqueous formic acid solution (80:20 v/v ratio); and
d) measuring the misoprostol acid content in the sample using a mass spectrophotometer as a detector device.
In this embodiment, an NSAID can optionally be added to the plasma.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a representative chromatogram showing a peak for 1500 pg/ml misoprostol acid in an aqueous solution.
Figure 2 is a representative chromatogram showing an interfering peak (shaded) in a blank plasma (containing CPDA as anticoagulant) without NSAID addition to separated plasma.
Figure 3 is a representative chromatogram showing a reduced interfering peak (shaded) height in a blank plasma (containing CPDA as anticoagulant) with an NSAID added to separated plasma.
Figure 4 is a representative chromatogram showing an interfering peak (shaded) in the blank plasma (containing EDTA as anticoagulant) without NSAID addition while collecting blood.
Figure 5 is a representative chromatogram showing minimized interference in blank plasma (containing EDTA as anticoagulant) with NSAID added while collecting blood.
DETAILED DESCRIPTION OF THE INVENTION
It has been noted during determination of misoprostol acid content in blood (including plasma and serum) that a high degree of analytical interference occurs, which could lead to inconsistent results. This analytical interference may be attributed to presence of endogenous PGE (or their congeners) in blood that can be extracted during the sample preparation procedures of the analytical method. Surprisingly it has been found that addition of a nonsteroidal antiinflammatory drug ("NSAID") to a biological fluid comprising a prostaglandin minimizes this interference to such an extent that the analytical method is acceptable to regulatory agencies.
The present invention relates to use of a NSAID during determination of prostaglandin content in a biological fluid.

In the context of the present invention, non-limiting examples of an NSAID include: salicylic acid and aspirin; acetic acid derivatives like diclofenac and aceclofenac; propionic acid derivatives like ibuprofen, naproxen, flurbiprofen, fenoprofen, ketoprofen, suprofen, fenbufen and fluprofen; acetic acid derivatives like tolmetin sodium, zomepirac, sulindac and indomethacin; fenamic acid derivatives like mefenamic acid and meclofenamate sodium; biphenylcarboxylic acid derivatives like diflunisal and flufenisal; oxicams like piroxicam, sudoxicam and. isoxicam; benzeneacetic acid derivatives like diclofenac; COX-2 inhibitors like valdecoxib, celecoxib and rofecoxib; and the like.
"Biological fluid," as used herein, refers to various aqueous liquids present in the body including blood, plasma, serum, breast milk, saliva, urine, and tissues. The terms "blood," "plasma," and "serum" will be used synonymously and interchangeably in the description of the invention.
In an embodiment, the present invention includes use of an NSAID for minimizing analytical interference during determination of prostaglandin content in a biological fluid.
Eicosenoids are 20-carbon fatty acids that are produced in a variety of tissues and biological fluids that mediate an array of physiologic and pathologic processes and are derived from oxidative metabolism of arachidonic acid. They consist of prostaglandin PGA through PGH, which are present in mammalian tissue where they regulate function.
Prostaglandins are classified as PGA, PGB, PGC, PGD, PGE, PGF, PGG and PGH based on their cyclopentane/pentane ring substitution patterns. Each general prostaglandin is sub-classified based on the degree of abbreviation indicates the nature of unsaturation and substitution.
The conversion of free arachidonic acid to prostaglandins and other eicosenoids is initiated by oxidative enzymes of the cyclooxygenase and lipoxygenase families.
Two different isozymes of cyclooxygenase exist, a constitutive form (COX-1) and a highly inducible form, (COX-2) .The cox-enzymes are variably inhibited by W3-fatty acids and nonsteroidal antiinflammatory drugs (NSAIDs).
In an embodiment, the present invention includes use of an NSAID for minimizing analytical interference during determination of prostaglandin content in a biological fluid.

Determination of any analyte content in biological fluid generally involves two steps: sample preparation and estimation of analyte content in the sample using a suitable analytical technique. Sample preparation comprises collection of biological fluid, optional pretreatments such as freezing and thawing, and extraction of analyte in suitable solvent system for further estimation.
In the context of the present invention, addition of an NSAID to biological fluid, particularly during sample preparation, has been found to minimize the analytical interference during estimation of prostaglandin content. In a specific embodiment, addition of a NSAID to a biological fluid during sample preparation has been found to minimize the analytical interference to such an extent that the level of analytical interference is less than about the lower limit of quantitation in a particular analytical method employed.
An NSAID can be used in the form of a solid powder or suspension or solution when added to biological fluid. Alternatively, a biological fluid can be collected in a container containing an NSAID in the form of a solid powder or suspension or solution, optionally together with other additives like an anticoagulant. Suitable vehicles including water, alcohols and other organic solvents, or mixtures thereof, can be employed for preparation of an NSAID suspension or solution, which can be mixed with biological fluid in the required proportions.
A specific embodiment of the present invention provides a method for determining the prostaglandin content in blood with minimized analytical interference, wherein said method comprises:
a) adding an NSAID to blood and separating the plasma; and
b) determining the prostaglandin content in plasma by a liquid chromatographic technique.
In another embodiment, the present invention provides use of an NSAID for minimizing analytical interference during a determination of misoprostol acid content in a biological fluid.
In another specific embodiment, the present invention provides a method for determining misoprostol acid content in blood with minimized analytical interference, wherein said method comprises:
a) separating the plasma from a blood sample;
b) adding an NSAID to the plasma; and

c) determining the misoprostol acid content in plasma by HPLC.
In a still further embodiment of the present invention, a high performance liquid chromatography technique for determination of misoprostol acid content in plasma includes:
a) extracting misoprostol acid from a plasma sample and dissolving it in a reconstitution solution comprising acetonitrile and a 0.2 % aqueous formic acid solution (80:20 v/v ratio);
b) injecting the sample solution onto an reversed phase ("RP") C18, 3.5 pm HPLC column;
c) eluting misoprostol acid from the column using a mobile phase comprising acetonitrile and a 0.2 % aqueous formic acid solution (80:20 v/v ratio); and
d) measuring the misoprostol acid content in the sample using a mass spectrophotometer as a detector device.
In a study that demonstrates use of the present invention, human subjects are administered a misoprostol-containing formulation, and at periodic intervals blood samples are collected in containers. An anticoagulant such as heparin, ethylenediamine tetraacetic acid ("EDTA") or a salts thereof like K3 EDTA or K2 EDTA, citrate phosphate dextrose adenine ("CPDA"), ammonium oxalate or potassium oxalate, and the like are often added to prevent clotting of blood samples, and the samples can then be stored under frozen conditions as required.
The concentration of an NSAID to be added to blood sample (or plasma separated from blood sample) may vary depending on the specific NSAID used. However any concentration of a NSAID that minimizes the analytical significance to acceptable levels will be useful in the context of the present invention. Determination of an appropriate amount is well within the ordinary skill in the art.
In an embodiment, an NSAID is added to a blood sample (or plasma separated from blood sample) in a concentration ranging from about 0.1 MQ/nnl to about 10 [jg/ml, or about 0.5 \iglrr\\ to about 5 (jg/ml. NSAID concentrations of about 1 jjg/ml of blood have been found to be particularly useful.
When required, the blood sample can be analyzed for misoprostol acid content using a suitable analytical technique including liquid chromatography ("LC"), gas chromatography ("GC"), thin layer chromatography ("TLC"), supercritical fluid chromatography ("SFC") and the like. Particularly, a high performance liquid

^.niu.naiuyrapny ^ rih-LU J technique was found to be suitable in the context of the present invention.
As is known to a person skilled in the art, various detector systems can be employed along with the above-mentioned analytical techniques. A few examples of suitable detectors include ultraviolet ("UV") detectors, fluorescence detectors, refractive index ("Rl") detectors, radiation detectors (for detecting radio-labeled compounds), and mass spectrometer ("MS") detectors. Particularly, a MS detector was found to be suitable in the context of the present invention.
In an aspect of the present invention, a HPLC technique coupled with tandem mass spectrometry (LC-MS/MS system) has been employed to determine misoprostol acid contents in blood.
Typically, determination of an analyte from a biological fluid involves extraction of the analyte into a suitable liquid using techniques including solid phase extraction, liquid-liquid extraction, direct precipitation, ultra centrifugation, etc. The extracted analyte, after suitable dilution, is then injected into a HPLC column (commercially available with different dimensions, stationary phases, and substrate sizes). As used herein, a RP C18, 3.5 pm HPLC column was found to be suitable in instant invention, although many other columns can also be used.
An embodiment of a sample preparation procedure, in the context of the present invention, comprises:
a) mixing a 1 ml plasma sample with 500 pi of 10 mM ammonium acetate buffer (pH 7.5 ) and 500 pi of water, and vortex mixing for 30 seconds; and
b) loading this mixture on a preconditioned solid phase extraction ("SPE") cartridge, washing the cartridge with 10 mM ammonium acetate buffer (pH 7.5), and finally eluting with mobile phase as described in the chromatography conditions.
In an aspect, the following typical experimental conditions are employed in a HPLC-MS/MS analytical technique for the determination of a misoprostol acid content in a biological fluid:
Mobile phase: 0.2% aqueous formic acid solution and acetonitrile in a ratio of 20:80 v/v.
HPLC column: Kromasil® CI8 (100x4.6 mm) 3.5 pm, Flexit®.
Injection volume: 50 pi.
Flow rate: 0.4 ml/minute.

Column oven temperature: about 40 °C.
Retention time: about 2.64 minutes (misoprostol acid).
MS scan type: Multiple Reaction Monitoring ("MRM").
Polarity: Negative.
Ion source used: Turbo spray.
Transitions monitored (Q1 mass): 367.1 amu, (Q3 mass): 249 amu.
The above-mentioned HPLC-MS/MS technique provides a lower limit of quantification ("LLOQ") of 8pg/mL. The LLOQ values could vary somewhat according to the analytical techniques that are employed for determination of an analyte.
It is to be understood that the above-mentioned HPLC-MS/MS technique is illustrative only and can be modified within its scope by a person skilled in the art so as to fulfill the analytical method requirements of precision, accuracy, sensitivity, selectivity, robustness, linearity and the like. Such other analytical techniques for determination of prostaglandins in biological fluids are within the purview of the present invention as long as the analytical interference is minimized by use of a NSAID during sample preparation as described in the context of the present invention.
The following examples illustrate certain specific aspects and embodiments of the invention and demonstrate the practice and advantages thereof. It is to be understood that the examples are given by way of illustration only and are not intended to limit the scope of the invention in any manner.
EXAMPLES EXAMPLE 1: Minimization of interference in misoprostol free acid assay by addition of diclofenac to human plasma.
A method has been developed to minimize interference in misoprostol free acid assay by addition of 1 [jg/ml of diclofenac to plasma. Anticoagulant used was a mixture of sodium citrate (dihydrate) 2.63 g, citric acid (monohydrate) 0.299 g, dextrose (monohydrate) 2.9 g, monobasic sodium biphosphate (monohydrate) 0.22 g and adenine 0.0275 g, per 100 ml of plasma.
1 ml of blank plasma with and without addition of diclofenac (1 jjg/ml) was taken for solid phase extraction. Plasma blank samples with and without diclofenac addition were processed in the manner described below:
1. 1 ml of plasma was taken into a vial. To this was added 0.5 ml of 10 mM
ammonium acetate (pH 7.5, adjusted with 50% ammonia solution, v/v) and

vortex mixed for 30 seconds. 0.5 ml of water was then added and vortex mixed for 30 seconds. This sample mixture was applied to solid phase extraction cartridges (Oasis™ MAX, 1 cc, 30 mg. Waters Corporation, Milford, Massachusetts USA). The cartridges were initially preconditioned with 1.0 ml of acetonitrile, followed by 1.0 ml of pure water.
2. The cartridges were washed with 1 ml of 10 mM ammonium acetate (pH 7.5, adjusted with 50% ammonia solution, v/v), then with 1 ml of water, and finally with 1 ml of 20% of acetonitrile in water, v/v.
3. The cartridges were dried under vacuum for 2 minutes and finally eluted with 0.5 ml of HPLC mobile phase (0.2% aqueous formic acid solution in acetonitrile (20:80 v/v ratio)
4. 50 |jl of eluent was injected into a Kromasil® CI8 (100 x 4.6 mm) 3.5 pm, Flexit® column, which was maintained at 40°C, and a flow rate of 0.4 ml per minute was maintained.
5. A tandem mass spectrometer was used for detection. Samples were analyzed in negative ion mode (polarity) and the scan type chosen was multiple reaction monitoring (MRM). Turbo spray was used as the source.
6. The following transitions were monitored for misoprostol acid: (Q1 mass): 367.1 amu (Q3 mass): 249.0 amu.
Retention time of about 2.41 minutes and about 2.23 minutes was observed for 1500 pg/ml misoprostol acid in plasma, and in aqueous solution, respectively.
The interfering peak, which eluted at 2.41 ±0.5 minutes for a plasma blank (without diclofenac) was minimized with addition of diclofenac in plasma.
Summary of retention times of interfering peak in 5 different sources of blank plasma samples, with and without diclofenac, is shown below.

s.
No. Retention Time (minutes) Area (counts) % Reduction in Area

Without Diclofenac With Diclofenac Without Diclofenac With Diclofenac

1 2.41 2.40 10176 9155 10.03
2 2.40 2.39 10977 5220 52.45
3 2.39 2.41 20633 9351 54.49
4 2.43 2.41 10416 6307 39.45

Figure 2 indicates blank plasma with higher interference, when diclofenac sodium was not added. As shown in Figure 3, when diclofenac sodium was added to blan(< plasma samples, the interference level was decreased to a significant extent. Thus, the interference level was decreased to significant extent by addition of diclofenac sodium to plasma.
EXAMPLE 2: Minimization of interference in misoprostol free acid assay by addition of diclofenac while collecting blood from humans.
A method has been developed to minimize interference in misoprostol free acid assay by addition of 1 [jg/ml of diclofenac in blood. Anticoagulant used was tripotassium EDTA (15% w/v).
Blood samples, with and without addition of diclofenac (1 |jg/ml), were centrifuged to separate plasma for further analysis. Further processing was the same as that described in Example 1 (steps 1 through 6).
The interfering peak, which eluted at 2.77±0.5 minutes in plasma (without diclofenac) was minimized when diclofenac was added to freshly collected blood.
Summary of retention times of the interfering peak in 3 different blood samples, with and without diclofenac, is shown below.
Retention Time (minutes) I Peak Area (counts/second)^
Without \ with Without I With
Diclofenac Diclofenac Diclofenac Diclofenac
2/77 Not Detected* T684 '-
2?77 Not Detected 2059 -
2J7 Not Detected 305 -
*Limit of detection 4 pg/ml.
Figure 4 (blood without diclofenac sodium) shows the interference. As shown in Figure 5, when diclofenac sodium was added while collecting blood and later separating the plasma, the interference was completely abolished to such a level that it was not detected (below limit of detection). This observation underscores the use of diclofenac for minimizing the interference during determination of misoprostol acid in plasma.

CLAIMS:
1. A method for determining prostaglandin drug content in a biological fluid,
comprising adding a nonsteroidal antiinflammatory drug to a sample of the fluid to be
analyzed.
2. The method of claim 1 wherein a prostaglandin drug comprises misoprostol.
3. The method of claims 1 or 2, wherein a nonsteroidal antiinflammatory drug comprises diclofenac or a salt thereof.
4. The method of any of claims 1-3, wherein a nonsteroidal antiinflammatory drug is added at a time of collecting a sample.
5. The method of any of claims 1-3, wherein a fluid is blood and a nonsteroidal antiinflammatory drug is added to separated plasma.
6. The method of any of claims 1-5, wherein an amount of an added nonsteroidal antiinflammatory drug is sufficient to minimize interference in an analysis caused by endogenous prostaglandins present in the biological fluid.
7. The method of claim 6, wherein about 0.1 to about 10 pg/ml of nonsteroidal antiinflammatory drug is added.
8. The method of claim 6, wherein about 0.5 to about 5 pg/ml of nonsteroidal antiinflammatory drug is added.
9. The method of claim 6, wherein about 1 pg/ml of nonsteroidal antiinflammatory drug is added.
10. The method of any of claims 1-9, wherein a sample is analyzed by high performance liquid chromatography or by high performance liquid chromatography tandem mass spectrometry.