Method for the Direct Detection and/or Quantification of at Least One Compound with a Molecular Weight of at Least 200

Abstract

The present invention relates to method for the direct detection and/or quantification of at least one compound with a molecular weight of at least 200, wherein the compound to be detected and/or quantified is a chemically complex molecule, wherein said chemically complex molecule is substituted with at least two groups R, wherein each R group means independently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with the proviso that at least two R are independently selected from —P(O)(OH)2 and —OP(O)(OH)2, wherein the compound or compounds to be detected and/or quantified are within a biological matrix, wherein said biological matrix is a biological fluid, a biological tissue, stomach contents, intestine contents, stool sample or a culture cells, wherein the method comprises performing a chromatography and identifying the retention time and/or the intensity of the signal by means of a mass or radioactivity detector.

Claims

1-20. (canceled)

21. A method for analyzing a composition comprising a phosphorus containing compound having a molecular weight of at least 200 daltons, wherein the method comprises (i) introducing at least one standard sample together or sequentially with a test sample comprising the composition comprising a phosphorus containing compound together with its impurities into a stream of a solvent system, wherein the solvent system is a polar solvent comprising potassium hydroxide (KOH) or a solvent mixture comprising at least one polar solvent comprising KOH, and, (ii) passing the standard sample and test sample through a single anion-exchange chromatography column containing particles of a cross-linked polystyrene resin, wherein the test sample comprises a solution or a slurry containing the composition comprising the phosphorus containing compound, and wherein the amount of phosphorus containing compound in the composition is above 60% by weight; wherein the phosphorus containing compound is selected from: (a) a bisphosphonate orpolyphosphonate; (b) a hexametaphosphate; (c) a C3-C7 cycloalkyl substituted compound with at least two —R groups wherein each —R group is —OH, —OP(O)(OH)2 or —P(O)(OH)2, and wherein at least two —R groups are independently selected from —P(O)(OH)2 and—OP(O)(OH)2 and said C3-C7 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl; (d) ions or salts thereof; or, (e) a combination thereof.

22. The method of claim 21, wherein the test sample has not undergone any sample pretreatment apart from diluting the test sample

23. The method of claim 21, comprising identifying the retention time and/or quantifying the intensity of the signal of the phosphorus containing compound when the standard sample and test sample are eluted from the anion-exchange chromatography column.

24. The method of claim 21, wherein the composition is selected from the group consisting of an active pharmaceutical ingredient (API), a drug, a medical food, a reagent, a food additive, a pharmaceutical composition and a nutraceutical composition.

25. The method of claim 21, wherein the polystyrene resin is cross-linked with divinylbenzene.

26. The method of claim 21, wherein the anion-exchange chromatography column is filled of the particles of cross-linked polystyrene resin as a stationary phase.

27. The method of claim 21, wherein the anion-exchange chromatography column is maintained at a pressure between 5 and 1500 atm.

28. The method of claim 23, wherein identifying the retention time and/or quantifying the intensity of the signal of the phosphorus containing compound comprises using a mass spectrometry detector.

29. The method of claim 23, wherein identifying the retention time and/or quantifying the intensity of the signal of the phosphorus containing compound comprises post column derivatization.

30. The method of claim 29, wherein the post column derivatization is followed by UV detection.

31. The method of claim 21, wherein the method further comprises quantifying the amount of the phosphorus containing compound in the composition.

32. The method of claim 21, wherein the method further comprises quantifying the purity of the phosphorus containing compound in the composition.

33. The method of claim 21, wherein the method is used to detect and quantify any of the impurities in an impurities profile.

34. The method of claim 21, wherein the C3-C7 cycloalkyl substituted compound is an inositol polyphosphate comprising from two to six phosphate groups.

35. The method of claim 34, wherein the inositol polyphosphate is phytate.

36. The method of claim 21, wherein the pH of the solvent system is between 7 and 14.

37. The method of claim 28, wherein the mass spectrometry detector is a tandem mass spectrometer, a triple quadrupole spectrometer, or a single quadrupole spectrometer.

38. The method of claim 23, wherein the mass spectrometry detector operates under selected ion monitoring (SIM) mode, multiple reaction monitoring (MRM) mode, selected reaction monitoring (SRM) mode, SCAN (positive/negative), or a combination thereof.

39. The method OF claim 21, wherein the standard sample is a reference standard, an external standard, an internal standard, or a standard addition.

40. A process for preparing a composition comprising a phosphorus containing compound having a molecular weight of at least 200 daltons, wherein the process comprises quantifying the compound together with its impurities, wherein the at least one phosphorus containing compound is selected from: i. a bisphosphonate orpolyphosphonate; ii. a hexametaphosphate; iii. a C3-C7 cycloalkyl substituted compound with at least two —R groups wherein each —R group is —OH, —OP(O)(OH)2 or —P(O)(OH)2, and wherein at least two —R groups are independently selected from —P(O)(OH)2 and—OP(O)(OH)2 and said C3-C7 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl; iv. ions or salts thereof; or, v. a combination thereof; and, wherein the process comprises measuring the percentage of purity of the phosphorus containing compound in the composition by a process comprising the method of claim 21, and including the batch of composition only if the percentage of purity of the phosphorus containing compound is above 60% by weight.

41. The process of claim 40, wherein the C3-C7 cycloalkyl substituted compound is an inositol polyphosphate comprising from two to six phosphate groups.

42. The process of claim 41, wherein the inositol polyphosphate is phytate.

43. The process of claim 40, wherein the compositions is an API, a drug, a medical food, a reagent, a food additive, a pharmaceutical composition, or a nutraceutical composition.

Description

DESCRIPTION OF THE FIGURES

[0130] FIG. 1. UPLC®-MS chromatogram obtained for IP6 in rat plasma after gradient-elution reversed-phase chromatography of example 1.

[0131] FIG. 2. HPLC-MS chromatogram obtained for IP6 in rat urine after gradient-elution reversed-phase chromatography of example 4.

[0132] FIGS. 3A and 3B. Typical Chromatogram of an API sample (detail in the case of the sample) of example 8 (impurities profile). FIG. 3A: Blank.

[0133] FIG. 3B: Sample (P: Phosphate, IP6: inositol hexaphosphate; 1, 2, 3 and 4: Related impurities).

[0134] FIG. 4. Typical Chromatogram of ATP for a plasma rat sample (Example 9).

[0135] FIG. 5. Typical Chromatogram of IP3 for a plasma rat sample (Example 10).

[0136] FIG. 6. Typical Chromatogram of IP6 in human urine (Example 11).

[0137] FIG. 7. Typical Chromatogram of IP6 using a solution of 30 mg BSA (Bovine Serum Albumin)/mL in PBS as a surrogate matrix for plasma or serum (Example 12).

[0138] FIG. 8. Typical Chromatogram of the impurities present in a Phytic Acid solution: IP5, IP4, IP3 and m/z 779 (Example 13)

[0139] FIG. 9. Typical Chromatogram of Phytic Acid metabolites m/z 779, m/z 740 y m/z 579 after incubation of Phytic Acid in rat hepatocytes (Example 14)

[0140] FIG. 10. Typical Chromatogram of Phytic Acid metabolites m/z 499, m/z 419, m/z 339 y m/z 259 after incubation of Phytic Acid in rat hepatocytes (Example 14)

[0141] FIG. 11. Typical Chromatogram of Phytic Acid after incubation of Phytic Acid in rat hepatocytes (Example 14)

[0142] FIG. 12. Typical Chromatogram of IP5 in rat plasma (Example 15)

[0143] FIG. 13. Typical Chromatogram of Phytic Acid metabolites (metabolic profile) in rat plasma (Example 16)

[0144] FIG. 14. Typical Chromatogram of Phytic Acid metabolites (metabolic profile) in dog plasma (Example 17)

[0145] FIG. 15. Typical Chromatogram of an API sample, showing the corresponding peaks for Phytic Acid and its related impurities (impurities profile) (Example 18)

[0146] The following examples illustrate the invention as disclosed herein and are not intended to limit the scope of the invention set forth in the claims appended thereto.

EXAMPLES

Example 1. Determination of IP6 in Rat Plasma Samples (SIM Mode)

[0147] The plasma sample underwent a purification and extraction of the compound by protein precipitation with TCA in presence of a chelating agent (EDTA). The supernatant was then diluted with triethylamine acetate (TEAA) and 20 μL was injected into UPLC®-MS system.

[0148] The ionization of phytic acid was assessed using negative electrospray ionization-mass spectrometry (ESI-MS).

[0149] Quantitative analysis was performed by mass spectrometry in the selected ion monitoring (SIM).

[0150] The compound was analyzed by gradient-elution reversed-phase chromatography using TEAA in aqueous solution and acetonitrile as mobile phase. The retention time of the analyte under the optimized chromatographic conditions is 3.42 min (see chromatogram FIG. 1)

Example 2. Determination of IP6 in Dog Plasma Samples (SIM Mode)

[0151] The bioanalytical procedure developed in Example 1, was revalidated in dog plasma samples with a full assessment of linearity, accuracy and precision. The calibration curve was developed by injecting 20 μl of blank plasma samples spiked with a known amount of IP6. Accuracy below 10% and precision below 15% at intermediate concentrations was obtained, resulting in an excellent bioanalytical method.

Example 3. Determination of IP6 in Human Plasma Samples (SIM Mode)

[0152] The bioanalytical procedure developed in Example 1 was applied in human plasma samples, but doubling the amounts of trichloroacetic acid (TCA), matrix and chelating agent. The retention time of the analyte under the optimized chromatographic conditions was 4.05 min.

Example 4. Determination of IP6 in Rat Urine Samples (SIM Mode)

[0153] The bioanalytical procedure involved an extraction of the compound by diluting the rat urine in presence of a chelating agent (EDTA) and no precipitating agent was needed. The supernatant was then diluted with triethylamine acetate (TEAA) and 50 μL was injected into the HPLC-MS system.

[0154] The ionization of Phytic acid was assessed using negative electrospray ionization-mass spectrometry (ESI-MS). Quantitative analysis was performed by mass spectrometry in the selected ion monitoring (SIM) mode.

[0155] The compound was analyzed by gradient-elution reversed-phase chromatography, using TEAA in aqueous solution and acetonitrile as mobile phase. The retention time of the analyte under the optimized chromatographic conditions was ˜3.99 min (see FIGS. 3A and 3B for the chromatogram)

Example 5. Determination of IP6 in Formulations (SIM Mode)

[0156] IP6 has been identified and quantified in formulations (solutions) of the active pharmaceutical ingredient. In this example, no simultaneous quantification of related impurities is performed. The solution media consisted of water, 0.9% NaCl or other aqueous solutions as vehicle.

[0157] The determination and quantification of IP6 was developed using the same procedure as in Example 1, without any sample pretreatment apart from diluting the formulation to fit within the calibration curve range.

[0158] The validated analytical method was also used to evaluate the stability as well as the homogeneity of IP6 in unfiltered formulations. The method was proved to be linear and specific.

Example 6. Determination of IP6 in Hepatocytes Culture Cells (SIM Mode)

[0159] The method described in Example 1 was successfully applied to hepatocytes culture cells. The study of the injection of several culture cells with a known amount of IP6 to the HPLC-MS showed a reliable method for this kind of matrix.

Example 7. Determination of IP6 in Pig Plasma Samples (SIM Mode)

[0160] The bioanalytical procedure developed in Example 1, was applied to pig plasma samples. The calibration curve was developed by injecting 50 μl of blank plasma samples spiked with a known amount of IP6. An accuracy and precision below 15% was obtained, resulting in an excellent bioanalytical method.

Example 8. Determination of IP6 and Related Impurities in Formulations

[0161] IP6 has been identified and quantified in formulations (solutions) of the active pharmaceutical ingredient. In this example, simultaneous determination of related impurities is performed, allowing the calculation of the chromatographic purity of the API.

[0162] The determination and quantification of IP6 was developed using potassium hydroxide was used as mobile phase. Sodium hydroxide can be alternatively used and the addition of small proportions of isopropanol is strongly recommended.

[0163] The used column was an anion-exchange divinyl benzene polymer. The flow rate was maintained at 1 mL/min with a temperature of 35° C.

[0164] Better sensitivity, especially for impurities, was obtained when using chemical or electrochemical ionic suppression.

[0165] The retention time for phytic acid was 24.6 minutes (see FIG. 4). The identity of IP6 is confirmed if the retention time of the main peak in the assay sample is within±0.5 minutes of the mean retention time of the peak corresponding to phytate for all injections of the assay working standard.

[0166] This technique allows the simultaneous determination of the API together with its related impurities in a single chromatogram run.

Example 9. Determination of ATP (Adenosine Triphosphate) in Rat Plasma Samples (SIM Mode)

[0167] The bioanalytical procedure developed in Example 1, was applied to rat plasma samples, but changing the monitored mass in the SIM mode with the molecular weigh (M-1) of ATP. The calibration curve was developed by injecting 50 μl of blank plasma samples spiked with a known amount of ATP. The retention time for ATP was 3.96 min. FIG. 4 shows a typical chromatogram obtained with these samples.

Example 10. Determination of IP3 (inositol triphosphate) in Rat Plasma Samples (SIM Mode)

[0168] The bioanalytical procedure developed in Example 1, was applied to rat plasma samples, but changing the monitored mass in the SIM mode with the molecular weight (M-1) of IP3. The calibration curve was developed by injecting 50 μl of blank plasma samples spiked with a known amount of IP3. The retention time for IP3 was 2.62 min. FIG. 5 shows a typical chromatogram obtained with these samples.

Example 11. Determination of IP6 (inositol hexaphosphate) in Human, Rat and Dog Plasma and in Human and Rat Urine (MRM Mode)

[0169] The bioanalytical method developed in Example 1 and used in example 1-10 was transferred to the MRM mode in an UPLCQMS/MS system. The use of this MRM mode resulted in an increase of the analytical sensitivity as well as selectivity improvement.

[0170] The same extraction procedure was used. Chromatographic conditions were also based on the same theory. The mass transition obtained after collision-induced dissociation and used for quantitative purpose of IP6 was m/z 659.0>m/z 560.9. FIG. 6 shows a typical chromatogram obtained with these samples.

Example 12. Determination of IP6 in a Surrogate Matrix (Serum or Plasma Surrogate Matrix)

[0171] The bioanalytical procedure developed in Example 11, was applied in a surrogate matrix. In a particular situation (e.g. constitutive levels of the analyte are expected in the blank matrix) a surrogate matrix could be used to prepare the calibration curve and to quantify IP6 in biological samples.

[0172] A similar matrix effect as well as the same extraction recovery between surrogate matrix and biological matrix was observed resulting in an identical behavior in the UPLC®-MS/MS system. As a surrogate matrix for plasma or serum, a solution of 30 mg BSA (Bovine Serum Albumin)/mL in PBS was used. This modification gives a higher sensitivity and avoids the use of natural biological matrices to build the calibration curve. FIG. 7 shows a typical chromatogram obtained with these samples.

Example 13. Determination of IP6 and Related Impurities in Formulations (SCAN Mode)

[0173] The chromatographic method developed for the determination of Phytic Acid permitted to determine some impurities presents in a Phytic Acid solution, all the detected impurities (IP3, IP4, IP5 and m/z 779) coeluted with the peak of Phytic Acid; however, they were detected due to their different molecular weight. IP5 was the most abundant impurity.

[0174] FIG. 8 shows a chromatogram obtained for this example.

Example 14. Determination of IP6 and Related Metabolites in Rat Hepatocytes (SCAN Mode)

[0175] Phytic Acid and its metabolites were detected after incubation of Phytic Acid in rat hepatocytes. The purification step involved a dilution of the pellets with KHB medium after precipitation with EDTA and finally a dilution with TEAA. Only IP1 and IP2 were eluted with significant differences in the retention time relative to Phytic Acid.

[0176] FIGS. 9, 10 and 11 show typical chromatograms of Phytic Acid and its metabolites after incubation of Phytic Acid in rat hepatocytes.

Example 15. Determination of IP5 in Dog and Rat Plasma Samples (SIM Mode)

[0177] The bioanalytical procedure developed in Example 1, was applied in the determination of IP5 in dog and rat plasma.

[0178] IP5 was detected in the SIM mode using molecular weight (M-1) of IP5. The retention time for IP5 was 4.10 min.

[0179] FIG. 12 shows a typical chromatogram obtained for IP5 in rat plasma.

Example 16. Determination of Phytic Acid Metabolites in Rat Plasma Samples (SIM Mode)

[0180] The bioanalytical procedure developed for the determination of Phytic Acid was applied to rat plasma samples in order to detect the maximum number of metabolites.

[0181] The metabolites were firstly detected in the SCAN mode and then confirmed and semi-quantified by SIM mode. Quantitative measurements can be performed obtaining (i.e. synthesizing) the corresponding metabolites to prepare standards. FIG. 13 shows a typical chromatogram obtained with these samples.

Example 17. Determination of Metabolites of Phytic Acid in Dog Plasma Samples (SIM Mode)

[0182] The bioanalytical procedure developed for the determination of Phytic Acid was applied to dog plasma samples in order to detect the maximum number of metabolites. The metabolites were firstly detected in the SCAN mode and then confirmed and semi-quantified by SIM mode. Quantitative measurements can be performed obtaining (i.e. synthesizing) the corresponding metabolites to prepare standards.

[0183] FIG. 14 shows a typical chromatogram obtained with these samples.

Example 18. Determination of phytic acid (Identity, Assay) and Related Impurities in Formulations for Quality Control of an API, a Medical Food, a Reagent, a Food Additive, a Pharmaceutical Composition or Nutraceutical

[0184] Phytic acid and related impurities have been identified and quantified in formulations in aqueous solution. This method allows the calculation of the assay and chromatographic purity of phytic acid.

[0185] The determination and quantification of phytic acid was carried out by Ion Chromatography with post column derivatization by UV detection.

[0186] The ion-chromatography column used was a polystyrene 2% cross-linked with divinylbenzene polymer. The flow rate was maintained at 1 mL/min, setting a column temperature of 35° C.

[0187] Adequate sensitivity for phytic acid related compounds was obtained by using post-column derivatization and UV detection.

[0188] The retention time for phytic acid was 48.06 minutes (see FIG. 15). The identity of phytic is confirmed when the retention time of the main peak in the assay sample is within±0.5 minutes of the mean retention time of the peak corresponding to phytic acid for all injections of the assay working standard.

[0189] This analytical technique allows the simultaneous determination of phytic acid together with its related impurities (up to thirty five) in a single chromatographic run for quality control of an API, a medical food, a reagent, a food additive, a pharmaceutical composition or nutraceutical.

[0190] Based upon tentative assignments from comparison of the relative retention times of the impurities formed with the literature method the major impurities formed are identified as listed in Table 1.

TABLE-US-00001 TABLE 1 Tentative impurity peak assignments for degraded sample Peak Retention Time (min) RRT DL-Ins (1, 5, 6) P3 17.400 0.36 — 21.533 0.45 — 22.467 0.47 DL-Ins (1, 2, 4, 6) P4 + 23.709 0.49 Ins (1, 2, 3, 5) P4 DL-Ins (1, 2, 3, 4) P4 + 24.733 0.51 Ins (1, 3, 4, 6) P4 DL-Ins (1, 2, 4, 5) P4 24.967 0.52 DL-Ins (1, 2, 4, 5) P4 27.233 0.57 DL-Ins (1, 2, 5, 6) P4 28.067 0.58 Ins (2, 4, 5, 6) P4 30.433 0.63 31.267 0.65 32.067 0.67 32.333 0.67 DL-Ins (1, 4, 5, 6) P4 33.467 0.70 Ins (1, 2, 3, 4, 6) P5 36.100 0.75 DL-Ins (1, 2, 3, 4, 5) P5 36.933 0.77 DL-Ins (1, 2, 4, 5, 6) P5 40.867 0.85 41.100 0.86 Ins (1, 3, 4, 5, 6) P5 42.067 0.88 42.300 0.88 Phytic Acid 48.067 1.00