The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-20 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention. With respect to claim 1, it is not clear if it is required to detect or quantify the one or more 11-oxygenated C19 steroids or if the claim is just requiring their presence in the sample. This is because the wherein statement that defines the one or more steroids to comprise one or more 11-oxygenated C19 steroids uses open language apparently covering detection or quantification of steroids other than 11-oxygenated C19 steroids. Examiner notes that claim 1 is a method for detecting or quantifying one or more steroids in a sample and step c) requires the detection or quantification of one or more steroids so that the open language of the wherein clause covers detection one or steroids where the steroids can be an 11-oxygenated C19 steroid or another steroid. Examiner also notes that claim 2 only further defines/limits what constitutes the 11-oxygenated C19 steroids but does not require that any of them are detected or quantitated. Because of the above observations and since claims 3 and 14 are directly dependent from claim 1 and includes steroids other than the 11-oxygenated C19 steroids that are common between them and claim 2, claim 1 is being treated as covering the detection or quantification of any steroid as long as the presence of the 11-oxygenated C19 steroids would have been expected in the sample by those of ordinary skill in the art. With respect to claim 7, it is not clear if the wash step with a wash solution having a pH of 2-4 is only required for certain types of SPE materials such as that required by claim 9 or is it SPE material independent. With respect to claims 8-9, “the solid phase” does not have antecedent basis in claim 1 but would if dependent from claim 6. Additionally, does the use of a magnetic particle change how the capturing, one or more optional wash steps and/or the eluting seps are performed? With specific reference to claim 9, it is not clear what form the SPE takes to be coated with the polymer. Is it the magnetic particles of claim 8, a glass (silica) or polymer particle or some other form coated with the required polymer? For examination purposes examiner will not limit the form of the polymer coated SPE. Claim 14 uses acronyms/abbreviations for the steroids being claimed. Those acronyms/abbreviations do not have antecedent basis in claim 1 but would if dependent from claim 3. For examining purposes claim 14 will be treated as if dependent from claim 3. Additionally claim 14 includes the language “is/are optionally used for quantification” which has clarity problems. From this language it appears that the quantitation is either optional and/or there is no requirement for the detected ions to be used to quantify the respective steroids. It is not clear which interpretation applicant intended. Because claim 1 is a “method for detecting or quantifying” examiner will treat the language as covering all possibilities: simply detecting one or more of the required fragment ions with no quantification, detecting one or more of the required fragment ions with quantification using an ion other than the one or more ions that are required to be detected or detecting one or more of the required ions and using the one or more required ions for quantitation. In claim 15, it is not clear if applicant is claiming that the whole method of only certain steps of the method are automated. Because page 32, lines 17-21 of the instant specification appears to teach that manual steps such as “the manual addition of a reagent to the sample and the transfer of the sample during processing from one device to another” are permitted (“Automated” means that except for the step of applying the sample and reagents to the system one or less, preferably no manual handling steps are required). For that reason, such manual steps are considered as within the scope of automated language of claim 15. Any claim not specifically addressed above is dependent from one or more of the above claims and fails to correct the issues of the claim(s) from which it depends.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-4, 6, 10-12, 14 and 18-20 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Draisci (Journal of High Resolution Chromatography 1997, newly cited and applied) in view of Schiffer (Annals of Clinical Biochemistry 2019, newly cited and applied). In the paper Draisci confirms anabolic hormone residues in bovine blood by micro-HPLC-Ion spray-Tandem mass spectrometry. With respect to claim 1, the paper teaches a method for detecting or quantifying one or more steroids (see at least the abstract, the determination of endogenous sex hormones: 17P-estradio1, progesterone and testosterone) in a sample using mass spectrometry, said method comprising: a) extracting the one or more steroids from the sample using solid phase extraction (SPE) so as to obtain an SPE extract comprising the one or more steroids (see at least section 2.4: aliquot (2 ml) of serum was spiked with 10 ng of 17-methyltestosterone (internal standard) and 15 ml of acetate buffer solutions (ABS) 0.15 M, pH 5, and the mixture was shaken on a vortex mixer. The sample was purified by solid phase extraction using a C18 cartridge); b) concentrating the one or more steroids, said concentrating comprising evaporating solvent from the SPE-extract obtained in a) ( see at least section 2.4: analytes were eluted with 4 ml of methanol, the solvent removed under nitrogen and the residue dissolved in 100 µ1 of methanol); and c) detecting or quantifying the one or more steroids in the sample using mass spectrometry (see at least section 2.5, figures 1 and 3-6 with their associated discussion and tables 1-2 with their associated discussion), wherein the one or more steroids comprise one or more 11-oxygenated C19 steroids (at least testosterone). Draisci does not teach the presence of 11-oxygenated C19 steroids in the sample.
In the paper Schiffer teaches that classical and 11-oxygenated androgens both contribute to the androgen pool. Regular monitoring of the androgen status is required in disorders of steroidogenesis, and multiplexing of androgens improves the diagnostic ability of an assay. Due to the cheap non-invasive collection, saliva is advantageous when multiple samples are required. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers sensitive, simultaneous quantification of steroids with short run times. They developed an LC-MS/MS assay for the simultaneous measurement of 17-hydroxyprogesterone, androstenedione, testosterone, 11ß-Hydroxyandrostenedione and 11-ketotestosterone in saliva (see figure 1 for the respective structures). The paragraph bridging pages 564-565 teaches that the determination of androgen status in female and male patients with disorders of steroidogenesis, in particular in congenital adrenal hyperplasia (CAH), is essential to assess the quality of their treatment and to stratify their androgen-related cardio-metabolic risk. However, adrenal and gonadal androgen biosyntheses are complex, and there is extensive peripheral activation of androgen precursors. Multiplexing of analytes is therefore required to improve the diagnostic ability of an androgen assay. The simultaneous measurement of the classical androgen testosterone and its precursor androstenedione in serum or saliva has previously been established as a sensitive marker for metabolic risk in polycystic ovary syndrome (PCOS), a syndrome of androgen excess in women. The first full paragraph on page 565 teaches that additionally, recent research on androgen biosynthesis pathways has identified the class of 11-oxygenated androgens as a significant driver of androgenic activity in addition to the classical androgens like testosterone. The 11-oxygenated androgen pathway is initiated by adrenal conversion of androstenedione to 11ß-Hydroxyandrostenedione (11OHA4), a major adrenal androgen precursor. 11OHA4 can be further converted to 11-ketotestosterone (11KT), which activates the androgen receptor with the same potency and efficacy as testosterone. In both common (PCOS) and rare (CAH) androgen excess conditions, 11-oxygenated androgens make the major contribution to the circulating androgen pool. It is therefore essential to include these analytes in the routine assessment of androgen status. Treatment monitoring in CAH requires regular and frequent sample collections and analysis. The collection of saliva for steroid analysis represents an excellent option for this, as it can be conducted by the patients on their own and at home, without the need for clinic attendance or support by clinical personnel. Additionally, a significant proportion of androgens in circulation is bound to sex-hormone binding globulin and therefore considered bio-inactive according to the free hormone hypothesis. Concentrations in saliva, however, represent the free, unbound fraction of the hormone and more likely reflect bioactive concentrations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is currently the tool of choice for steroid analysis due to its outstanding selectivity and short run times. Additionally, LC-MS/MS has the sensitivity to accurately quantify low analyte concentrations, as are expected in saliva, and allows for multiplexing of analytes of interest. Table 1 teaches that for 17-hydroxyprogesterone (17OHP) the parent ion has an m/z value of 331.2 and the fragment ions have m/z values of 97.1 and 109.1. Table 1 also teaches that for androstenedione (A4) the parent ion has an m/z value of 287.15 and the fragment ions have m/z values of 97.1 and 109.1. Table 1 also teaches that for testosterone (T) the parent ion has an m/z value of 289.1 and the fragment ions have m/z values of 97.0 and 109.0. Table 1 also teaches that for 11ß-Hydroxyandrostenedione (11OHA4) the parent ion has an m/z value of 302.9 and the fragment ions have m/z values of 97.0 and 145.0. Table 1 also teaches that for 11-ketotestosterone (11KT) the parent ion has an m/z value of 302.9 and the fragment ions have m/z values of 121.1 and 259.1. The online solid phase extraction and liquid chromatography section on page 567 teaches solid-phase extraction of the analytes using a C18 cartridge conditioned with methanol and water. The cartridge was washed with 30% (v/v) methanol in water and subsequently eluted using 55% of mobile phase A (2 mmol/L ammonium acetate in 0.1% (v/v) formic acid in distilled Milli-Q water) and 45% of mobile phase B (2 mmol/L ammonium acetate in 0.1% formic acid in ultra-pure methanol). A Waters TQ-S mass spectrometer was used for quantification. Table 1 precursor and product ions used for quantitation of the androgens.
Based on the teachings of Schiffer, one of ordinary skill in the art would have expected the sample of Draisci to comprise one or more 11-oxygenated C19 steroids.
With respect to claim 2, based on the teachings of Schiffer, one of ordinary skill in the art would have expected the one or more 11-oxygenated C19 steroids to be selected from the group consisting of 11ß-Hydroxyandrostenedione (11-OHA4), 11-Ketotestosterone (11KT), 11-Ketoandrostenedione (11KA4), and 11ß-Hydroxytestosterone (110HT). With respect to claim 3, the one or more steroids comprise testosterone (see at least the abstract, figures 1 and 3-6 with their associated discussion and tables 1-2 with their associated discussion). With respect to claim 4, section 2.4 teaches that the sample is serum or plasma. With respect to claim 6, section 2.4 teaches that the SPE comprises: d) capturing the one or more steroids to a solid phase (the sample was purified by solid phase extraction using a C 18 cartridge); e) optionally one or more wash steps (After sample loading, the cartridge was washed with 5 ml of ABS, 10 ml of water, 2.5 ml of methanol-water, 40:60 (v/v)); and f) eluting the one or more steroids from the solid phase (section 2.4: analytes were eluted with 4 ml of methanol, the solvent removed under nitrogen and the residue dissolved in 100 p1 of methanol). With respect to claim 10, evaporating of the solvent of the SPE extract results in a liquid volume reduction of 50 to 100% (section 2.4: analytes were eluted with 4 ml of methanol, the solvent removed under nitrogen and the residue dissolved in 100 p1 of methanol). With respect to claim 11, the mass spectrometry is a LC-MS (see at least the title and section 2.5). With respect to claim 12, the liquid chromatography (LC) is HPLC (see at least the title and section 2.5). With respect to claim 14, section 2.5 teaches that the one or more steroids comprise testosterone (T), and wherein for T a positively charged parent ion having an m/z value of 289.2 ± 0.5 is generated and selected for fragmentation, and wherein a first positively charged T-fragment ion having an m/z ratio of 97.1 ± 0.5 and/or a second positively charged T-fragment ion having an m/z ratio of 109.1 ± 0.5 are generated by fragmentation of the parent ion selected for T, and wherein said first and/or second T-fragment ion is/are detected by the mass spectrometry and is/are optionally used for quantification (see the mass spectra of testosterone in figures 1 and 3). With respect to claims 18-20, section 2.5 of the paper teaches that the mass spectrometry is a LC-MS/MS analysis and the liquid chromatography (LC) is reversed phase on a C18 column.
Claims 1-6, 10-14, 16 and 18-20 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Karvaly (Journal of Pharmaceutical and Biomedical Analysis 2018, newly cited and applied) in view of the teachings of Schiffer as described above. In the paper Karvaly provides a comprehensive characterization of adrenocortical steroidogenesis using two-dimensional ultra-performance liquid chromatography – electrospray ionization tandem mass spectrometry. With respect to claim 1, the paper teaches a method for detecting or quantifying one or more steroids (see at least the abstract, evaluating the serum levels of all respective major substances - aldosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dihydrotestosterone, 17a-hydroxypregnenolone, 17a-hydroxyprogesterone, corticosterone, cortisol, cortisone, pregnenolone, progesterone and testosterone) in a sample using mass spectrometry, said method comprising: a) extracting the one or more steroids from the sample using solid phase extraction (SPE) so as to obtain an SPE extract comprising the one or more steroids (see at least the abstract and section 2.3: 0.2 mL serum sample was mixed with 0.05 mL internal standard solution. 0.6 mL ice-cold methanol was added, followed by brief vortexing and centrifugation at 13000g for 5 minutes. 0.65 mL supernatant was separated and diluted with 1.3 mL water. Solid phase extraction was carried out on Phenomenex StrataTM-X 33 µm, 60 mg, 2 mL polymeric reversed phase 96-well plates. The phase was conditioned with 1.95 mL methanol followed by 1.95 mL water-methanol (3:1, v/v). The diluted sample supernatants were applied and allowed to drip through. The wells were washed 3 times with 1.95 mL water-methanol (3:1, v/v). After drying the wells under full vacuum for 2 min, the analytes were eluted by applying 2 × 0.85 mL acetonitrile-methanol (1:1, v/v). The eluates were evaporated to dryness, reconstituted with 0.045 mL methanol-water (1:1, v/v) and submitted for analysis); b) concentrating the one or more steroids, said concentrating comprising evaporating solvent from the SPE-extract obtained in a) ( see at least section 2.3: the analytes were eluted by applying 2 × 0.85 mL acetonitrile-methanol (1:1, v/v). The eluates were evaporated to dryness, reconstituted with 0.045 mL methanol-water (1:1, v/v) and submitted for analysis); and c) detecting or quantifying the one or more steroids in the sample using mass spectrometry (see at least section 2.4, figures 2-3 with their associated discussion and tables 1-4 with their associated discussion), wherein the one or more steroids comprise one or more 11-oxygenated C19 steroids (at least dehydroepiandrosterone and testosterone). Karvaly does not teach the presence of 11-oxygenated C19 steroids in the sample.
Based on the teachings of Schiffer, one of ordinary skill in the art would have expected the sample of Karvaly to comprise one or more 11-oxygenated C19 steroids.
With respect to claim 2, based on the teachings of Schiffer, one of ordinary skill in the art would have expected the one or more 11-oxygenated C19 steroids to be selected from the group consisting of 11ß-Hydroxyandrostenedione (11-OHA4), 11-Ketotestosterone (11KT), 11-Ketoandrostenedione (11KA4), and 11ß-Hydroxytestosterone (110HT). With respect to claim 3, the one or more steroids comprise testosterone (see at least the abstract, figures 1 and 3-6 with their associated discussion and tables 1-2 with their associated discussion). With respect to claim 3, the one or more steroids comprise dehydroepiandrosterone and testosterone (see at least the abstract, figures 2-3 with their associated discussion and tables 1-4 with their associated discussion). With respect to claim 4, section 2.3 teaches that the sample is serum or plasma. With respect to claims 5 and 16, section 2.3 teaches that the sample volume subjected to the SPE in a) is less than 500 µ1, less than 250 µ1 or less than 200 µl (0.2 mL (200 µl) serum sample was mixed with 0.05 mL internal standard solution. 0.6 mL ice-cold methanol was added). With respect to claim 6, section 2.3 teaches that the SPE comprises: d) capturing the one or more steroids to a solid phase (Solid phase extraction was carried out on Phenomenex StrataTM-X 33 µm, 60 mg, 2 mL polymeric reversed phase 96-well plates); e) optionally one or more wash steps (wells were washed 3 times with 1.95 mL water-methanol (3:1, v/v)); and f) eluting the one or more steroids from the solid phase (section 2.3: the analytes were eluted by applying 2 × 0.85 mL acetonitrile-methanol (1:1, v/v)). With respect to claim 10, evaporating of the solvent of the SPE extract results in a liquid volume reduction of 50 to 100% (section 2.3: analytes were eluted by applying 2 × 0.85 mL acetonitrile-methanol (1:1, v/v). The eluates were evaporated to dryness, reconstituted with 0.045 mL methanol-water (1:1, v/v) and submitted for analysis). With respect to claim 11, the mass spectrometry is a LC-MS (see at least the title and section 2.4). With respect to claim 12, the liquid chromatography (LC) is HPLC (see at least the title and section 2.4). With respect to claim 13, section 2.4 teaches that the LC is a RP-HPLC and the RP-HPLC comprises a gradient elution (a Phenomenex Kinetex XB-C18 to a Phenomenex Kinetex Biphenyl (both 50 × 2.1 mm, particle size: 1.7 µm) analytical columns, thermostated at 40 °C. The mobile phase consisted of water-formic acid (99.9:0.1, v/v, A) and methanol-formic acid (99.9:0.1, v/v, B). The following gradient program was used (% B): initial, 60%, 1.00 min, 60%, linear ramp to 4.00 min, 100%, 4.50 min, 100%, 4.51 min, 60%. The run time was 5.50 min, the cycle time was approximately 7.5 min, and the injection volume was 5.0 µL ). With respect to claim 14, table 1 teaches that the one or more steroids comprise testosterone (T), and wherein for T a positively charged parent ion having an m/z value of 289.2 ± 0.5 is generated and selected for fragmentation, and wherein a first positively charged T-fragment ion having an m/z ratio of 97.1 ± 0.5 and/or a second positively charged T-fragment ion having an m/z ratio of 109.1 ± 0.5 are generated by fragmentation of the parent ion selected for T, and wherein said first and/or second T-fragment ion is/are detected by the mass spectrometry and is/are optionally used for quantification (see spectra of testosterone (T) in figure 2 and the information provided in table 1). With respect to claims 18-20, section 2.4 of the paper teaches that the mass spectrometry is a LC-MS/MS analysis and the liquid chromatography (LC) is reversed phase on a C18 column (Phenomenex Kinetex XB-C18).
Claims 1-6, 10-14, 16 and 18-20 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Ponzetto (Analytical and Bioanalytical Chemistry 2016, newly cited and applied) in view of Schiffer as described above. In the paper Ponzetto describes longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports. With respect to claim 1, the paper teaches a method for detecting or quantifying one or more steroids (see at least the abstract and the compounds listed in table 1: testosterone, epitestosterone, androstenedione, DHEA (dehydroepiandrosterone) and DHT (dihydrotestosterone) in a sample using mass spectrometry, said method comprising: a) extracting the one or more steroids from the sample using solid phase extraction (SPE) so as to obtain an SPE extract comprising the one or more steroids (see at least the sample preparation section on page 707: Supported liquid extraction (SLE) on ISOLUTE SLE+ 400 μL 96-well plates was used to extract steroid hormones from serum samples. For each sample, 200 μL of serum were spiked with 20 μL of the IS-mix, diluted with 200 μL of water, and then agitated for 15 min at 250 rpm. Each well was then loaded with 400 μL of each serum sample and positive pressure of 3 psi was applied for 30 s to facilitate sample loading and adsorption; the elution was carried out, after a 5 min waiting period, by adding 700 μL of DCM to each well and applying a pressure of 3 psi for 1 min. Extracts were collected in collection plates equipped with 1.5 mL glass inserts, evaporated for approximately 15 min at 40 °C under a stream of nitrogen, and finally reconstituted with 100 μL of aMeOH-H2O 50:50 (v/v) solution (reconstitution solvent). After 15 min of gentle shaking (250 rpm), 10 μL of each extract were injected into UHPLCMS/MS for analyses); b) concentrating the one or more steroids, said concentrating comprising evaporating solvent from the SPE-extract obtained in a) (see at least the sample preparation section on page 707: the elution was carried out, after a 5 min waiting period, by adding 700 μL of DCM to each well and applying a pressure of 3 psi for 1 min. Extracts were collected in collection plates equipped with 1.5 mL glass inserts, evaporated for approximately 15 min at 40 °C under a stream of nitrogen, and finally reconstituted with 100 μL of aMeOH-H2O 50:50 (v/v) solution (reconstitution solvent). After 15 min of gentle shaking (250 rpm), 10 μL of each extract were injected into UHPLCMS/MS for analyses); and c) detecting or quantifying the one or more steroids in the sample using mass spectrometry (see at least the androgens, progestogens, and corticoids analysis section on page 707, and table 1 with its associated discussion), wherein the one or more steroids comprise one or more 11-oxygenated C19 steroids (at least testosterone, epitestosterone, androstenedione, DHEA (dehydroepiandrosterone )). Ponzetto does not teach the presence of 11-oxygenated C19 steroids in the sample.
Based on the teachings of Schiffer, one of ordinary skill in the art would have expected the sample of Ponzetto to comprise one or more 11-oxygenated C19 steroids.
With respect to claim 2, based on the teachings of Schiffer, one of ordinary skill in the art would have expected the one or more 11-oxygenated C19 steroids to be selected from the group consisting of 11ß-Hydroxyandrostenedione (11-OHA4), 11-Ketotestosterone (11KT), 11-Ketoandrostenedione (11KA4), and 11ß-Hydroxytestosterone (110HT). With respect to claim 3, the one or more steroids comprise testosterone, epitestosterone, androstenedione and DHEA (dehydroepiandrosterone) (see at least table 1 with its associated discussion). With respect to claim 4, the sample preparation section on page 707 teaches that the sample is serum or plasma. With respect to claims 5 and 16, the sample preparation section on page 707 teaches that the sample volume subjected to the SPE in a) is less than 500 µ1, less than 250 µ1 or less than 200 µl (200 µl serum sample spiked with 20 μL of the IS-mix, diluted with 200 μL of water). With respect to claim 6, the preparation section on page 707 teaches that the SPE comprises: d) capturing the one or more steroids to a solid phase (Supported liquid extraction (SLE) on ISOLUTE SLE+ 400 μL 96-well plates); e) optionally one or more wash steps (no washing steps but since the step is optional the lack of a washing step does not prevent anticipation); and f) eluting the one or more steroids from the solid phase (see at least the sample preparation section on page 707: the elution was carried out, after a 5 min waiting period, by adding 700 μL of DCM to each well and applying a pressure of 3 psi for 1 min). With respect to claim 10, evaporating of the solvent of the SPE extract results in a liquid volume reduction of 50 to 100% (see at least the sample preparation section on page 707: Extracts were collected in collection plates equipped with 1.5 mL glass inserts, evaporated for approximately 15 min at 40 °C under a stream of nitrogen, and finally reconstituted with 100 μL of aMeOH-H2O 50:50 (v/v) solution). The eluates were evaporated to dryness, reconstituted with 0.045 mL methanol-water (1:1, v/v) and submitted for analysis). With respect to claim 11, the mass spectrometry is a LC-MS (see at least the abstract and androgens, progestogens, and corticoids analysis section on page 707). With respect to claim 12, the liquid chromatography (LC) is HPLC (see at least the abstract and androgens, progestogens, and corticoids analysis section on page 707). With respect to claim 13, androgens, progestogens, and corticoids analysis section on page 707 teaches that the LC is a RP-HPLC and the RP-HPLC comprises a gradient elution. With respect to claim 14, table 1 teaches that the one or more steroids comprise testosterone, epitestosterone, androstenedione and DHEA (dehydroepiandrosterone ) with the respective precursor ions and at least one product ion for each of these analytes. With respect to claims 18-20, see at least the abstract and androgens, progestogens, and corticoids analysis section on page 707 of the paper teaches that the mass spectrometry is a LC-MS/MS analysis and the liquid chromatography (LC) is reversed phase on a C18 column (an Ethylene Bridged Hybrid (BEH) C18 column).
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Draisci, Karvaly or Ponzetto in view of Schiffer as applied to claim 10 above, and further in view of Pichon (Journal of Chromatography A 2000, newly cited and applied) or Miossec (Journal of Separation Science 2019, newly cited and applied). With respect to claim 17, Draisci, Karvaly or Ponzetto do not teach evaporating of the solvent of the SPE extract results in a liquid volume reduction of 60 to 85%.
In the paper Pichon reviewed solid-phase extraction for multiresidue analysis of organic contaminants in water. To overcome the limitations of the detection systems associated with gas or liquid chromatography, a sample pretreatment is required with the objective to provide a sample fraction enriched with all the target analytes and as free as possible from other matrix components. There is now no doubt that solid-phase extraction (SPE) has now become the method of choice for carrying out simultaneously the extraction and concentration of many compounds in aqueous samples. Many recent applications of SPE to multiresidue analysis were reviewed with an emphasis on the importance of the choice of the sorbent and of the sample volume. SPE is particularly well adapted to multiresidue analysis including compounds from a wide range of polarity or characterized by various physico–chemical properties. However, SPE is not completely free from practical problems inherent to the nature of the compounds or to the coupling to the chromatographic systems. Many examples are reported to illustrate these problems which can in most cases be circumvented. New developments in SPE were also reviewed. The second paragraph on page 196 teaches that problems can result from the loss of volatile compounds during the evaporation step or from the reconstitution of dry extracts containing compounds with large differences in water solubility. Section 4 goes over the choice of different sorbents to use in the extraction process such as n-alkyl bonded silicas (section 4.1), apolar copolymers (section 4.2) and/or carbonaceous sorbents (section 4.3). Section 5 covers extraction of analytes over a wide range of polarity in one run. Relevant to the instant claim is the last full paragraph on page 205 teaching that in order to limit the loss of volatile compounds, the evaporation step is carried out under mild conditions, i.e., under a stream of nitrogen instead of the use of a rotary evaporator until a final volume of 50–500 µl and a fraction of this extract is injected for analysis. When dry extracts are obtained, it is sometimes difficult to solubilize the residue. For LC analysis, it is recommended to use a solvent corresponding to the composition of the mobile phase at the beginning of the gradient. However, when very polar and non-polar analytes are present together, a complete solubilization of the extract can be impossible: an addition of water is required for the more polar ones, whereas very hydrophobic analytes can only be dissolved using non-polar organic solvents.
In the paper Miossec analyzed the multi-residues of 44 pharmaceutical compounds in environmental water samples by solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry. A solid-phase extraction combined with a liquid chromatography-tandem mass spectrometry analysis has been developed and validated for the simultaneous determination of 44 pharmaceuticals belonging to different therapeutic classes (i.e., antibiotics, anti-inflammatories, cardiovascular agents, hormones, neuroleptics, and anxiolytics) in water samples. The sample preparation was optimized by studying target compounds retrieval after the following processes: i) water filtration, ii) solid phase extraction using Waters Oasis HLB cartridges at various pH, and iii) several evaporation techniques. The method was then validated by the analysis of spiked estuarine waters and wastewaters before and after treatment. Analytical performance was evaluated in terms of linearity, accuracy, precision, detection, and quantification limits. Recoveries of the pharmaceuticals were acceptable, instrumental detection limits varied between 0.001 and 25 pg injected and method quantification limits ranged from 0.01 to 30.3 ng/L. The precision of the method, calculated as relative standard deviation, ranged from 0.3 to 49.4%. This procedure has been successfully applied to the determination of the target analytes in estuarine waters and wastewaters. Eight of these 44 pharmaceuticals were detected in estuarine water, while 26 of them were detected in wastewater effluent. As expected, the highest values of occurrence and concentration were found in wastewater influent. The final SPE procedure is given in section 2.4. The final LC procedure is given in section 2.5. The final MS/MS procedure is given in section 2.6. Section 3.1 discusses the steps taken to optimize the sample presentation. Particularly relevant to claim 17 is section 3.1.3 related to the evaporation step. This section teaches that evaporation could be a critical step, especially for the most volatile compounds which may be degraded. At that point, different evaporation conditions had been used for the evaporation of pharmaceutical compounds: evaporation to dryness under nitrogen stream, evaporation to dryness under nitrogen stream at 40 °C, and partial evaporation under nitrogen stream. In order to investigate retrievals of compounds after evaporation, 8 mL of a solution containing all compounds in MeOH was evaporated under three different conditions: to dryness under a gentle air stream and reconstitution with 1 mL of water/MeOH (75/25 v/v), to dryness under a gentle air stream at 40 °C, and reconstitution with 1 mL of water/MeOH (75/25 v/v), under a gentle air stream and evaporation stopped when approximately 250 μL of solution remained in the tube and then adjusted to 1 mL with water. Areas of target molecules were compared to areas obtained for a non-evaporated solution (Figure 4). Results showed that all compounds had the same overall behavior and resisted well to evaporation (recoveries above 70% for 84% of the target compounds). A significant loss was noticed for some antibiotics (tetracycline, doxycycline, josamycin, rifampicin), whatever the evaporation technique was. As there were no significant differences between these different techniques, evaporation to dryness under air stream at room temperature was chosen as a gentle evaporation method in order to concentrate while preserving molecules as much as possible. Losses observed for some analytes were subsequently bypassed by the quantification with an internal procedural calibration. Examiner notes that while evaporation to dryness at 40 °C produced the highest recovery for most compounds the best recovery for tetracycline and doxycycline was the partial evaporation process and the best recovery for oxolinic acid was evaporation to dryness. Section 4 suggests that the analytical method could be adapted to other matrices, such as biological tissues and sediments for evaluating the environmental quality of aquatic ecosystems.
With respect to claim 17, it would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Draisci, Karvaly or Ponzetto methods to reduce the volume of the eluant from the SPE to a volume of 50–500 µl as taught by Pichon or examine the evaporation methods to determine the best method for the measured analytes as taught by Miossec because of the expectation/possibility that it is sometimes difficult to solubilize the residue as taught by Pichon or the expectation that different evaporation methods may affect or be critical to the recovery of the analyte as taught by Miossec and the 100 µl volume of the reconstituted Draisci and Ponzetto solutions used for LC-MS analysis.
Claims 2 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Draisci, Karvaly or Ponzetto in view of Schiffer as applied to claims 1-2 above. Draisci, Karvaly or Ponzetto do not teach detection of quantification of the claimed 11-oxygenated C19 steroids.
It would have been obvious to one of ordinary skill in the art at the time the application was filed to include the 11-oxygenated androgens of Schiffer into the analyses of Draisci, Karvaly or Ponzetto because of the ability to improve the diagnostic ability of an assay by multiplexing the number and type of androgens as taught by Schiffer and the similarity in the assays used to measure the steroid analytes.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Draisci, Karvaly or Ponzetto in view of Schiffer as applied to claim 1 above, and further in view of Huq (Application note 2008, newly cited and applied). Draisci, Karvaly or Ponzetto do not specifically teach an automated SPE extraction.
In the application note Huq teaches that neutral sorbents are preferred for the solid-phase extraction of testosterone, a hydrophobic anabolic steroid, since these maximize retention due to strong hydrophobic interactions. However, elimination of contaminants from plasma samples requires a strong organic wash, which also weakens and disrupts such hydrophobic interactions. By providing additional interactive features such as π-π and hydrogen bonding interactions, Strata™-X enhances the retention of testosterone and sustains a 70 % methanol wash. In contrast, the silica-based Strata® C18-E can tolerate only a 40 % methanol wash without loss of analyte. Such a wash is necessary to eliminate phospholipids and minimize ion suppression and other matrix effects, thereby improving analytical accuracy and precision. Hormones belong to the steroid family and are chemical messengers produced by the endocrine glands, which directly release them into the bloodstream for transportation to various body parts. Examples of hormones include androgens, estrogens, progesterone and corticosteroids. Hormones are crucial for body function ranging from anti-inflammatory effects to regulation of events during pregnancy. Defects in steroid metabolism lead to diseases such as cancer, diabetes, cartilage/bone damage and neurological problems. For the qualitative/quantitative analysis of testosterone in biological matrices, both GC/MS and LC/MS are commonly employed. In either case, sample preparation is essential to remove the proteins and other endogenous/exogenous materials from whole blood, serum, plasma, urine or tissue samples. Solid-phase extraction (SPE) is the most selective and popular method of sample clean-up and is used in the off-line tube or 96-well plate format; a few examples of dispersive SPE have also been reported. Silica-based neutral (C18) or mixed mode (cation exchange/C8) sorbents, as well as polar polymeric sorbents have been used for SPE of testosterone and other steroids in biological matrices. However, a major problem with these methods is the low organic wash which does not effectively remove contaminants. In this technical note, they present an automated SPE protocol based on the neutral polar polymeric SPE sorbent, Strata-X, that furnishes ultra-clean samples from plasma matrices. For comparison, results from a C18-E (hydrophobically end-capped) silica sorbent were also presented. It is inferred that this method will be applicable to steroids and hormones in general, or other similar small molecules in a complex biological matrix. The automated SPE procedure was performed on a PerkinElmer® MultiPROBE II® automated liquid handler using a 96-well plate vacuum manifold deck accessory. Human plasma (500 μL), spiked with testosterone, was diluted with 1 mL of HPLC grade water and treated according to the steps listed in the last paragraph on the first page of the reference. The pooled eluent was evaporated to dryness under nitrogen and reconstituted in 500 μL of mobile phase prior to HPLC analysis. The analysis was performed using an HP 1100 LC system from Agilent Technologies equipped with a quaternary pump, in-line degasser, multi-wavelength detector and autosampler. The conclusion at the end of the reference is that an automated protocol incorporating a strong organic wash for the solid-phase extraction clean-up of testosterone from plasma employing Strata-X has been demonstrated to furnish very clean extracts with good recovery yields.
It would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the methods of Draisci, Karvaly or Ponzetto by using an automated SPE system of in combination with an autosampler type of liquid chromatography/mass spectrometry system as taught by Huq because such systems are known as shown by Huq and would be expected to reduce manual/operator performed steps.
Claim 8 is rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Draisci in view of Schiffer as applied to claim 1 above, and further in view of Liu (Microchemical Journal 2008, newly cited and applied). Draisci does not teach that magnetic particles are the solid phase used for the SPE.
In the paper Liu teaches the analysis of estrogens in water by magnetic octadecylsilane particle extraction prior to sweeping micellar electrokinetic chromatography. A method based on magnetic separation was developed for the extraction of several estrogens (including diethylstilbestrol, estrone and estriol) in water followed by sweeping micellar electrokinetic chromatography (MEKC) analysis with UV detection. Novel magnetic octadecylsilane
(ODS) particles were prepared using a silanization method with octadecyl trimethoxysilane as the surface modification reagent of magnetic Fe3O4 particles. Octadecyl trimethoxysilane was covalently immobilized on the magnetic iron oxide particles. The particles were used as the sorbents in the magnetic separation for the extraction of trace amounts of estrogens from water. The extraction condition and efficiency of the particles for the estrogens were investigated. Combining the magnetic ODS particles extraction and sweeping MEKC with UV detection, the estrogens at concentrations as low as ng/mL in water can be detected without interference from other substances in the sample matrix. Section 2.5 presents the magnetic extraction procedure. Magnetic ODS particles (0.1 g) were preconditioned with 10 mL of methanol, followed by 10 mL of ultra-pure water before use. After stirring, the sample solution (10 mL) was added to a 15 mL glass tube containing the magnetic particles. The mixture was vibrated gently for 1 h. Then, the particles were aggregated using a magnetic separator and the solution was removed. The particles were rinsed three times with water. Methanol (5 mL) was used to desorb the adsorbed estrogens on the particles. The methanol solution was evaporated using a micro centrifugal vacuum concentrator at 30 °C. Prior to analysis, the dried sample was reconstituted in 0.5 mL of 50 mM phosphoric acid, and then filtered with 0.45 μm cellulose acetate filter. The conclusion teaches that the results of the paper demonstrated the analytical procedure can eliminate matrix interferences and enhance the detection sensitivity.
It would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the extraction procedure of Draisci to use a magnetic particle C18 solid-phase such as the magnetic ODS particles of Liu because of the ability to separate steroids from samples to eliminate matrix affect and enhance the detection sensitivity as taught by Liu.
Claim 7 is rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Draisci in view of Schiffer as applied to claim 1 above, and further in view of Abdel-Khalik (Journal of Chromatography B 2013, newly cited and applied). Draisci does not teach that the one or more wash steps use a wash solution having a pH of 2 to 4.
In the paper Abdel-Khalik teaches of a solid phase extraction method for the simultaneous determination of steroid hormones in H295R cell line using liquid chromatography–tandem mass spectrometry. The H295R in vitro cell line produces the majority of the steroidogenesis, for which reason it is commonly used as a screening tool for endocrine disrupting chemicals. Simultaneous determination of the precursor cholesterol and key steroid hormones could give a broad insight into the mechanistic disruption of the steroidogenesis. Steroid hormones have primarily been extracted from H295R incubation medium by means of liquid–liquid extraction (LLE) and the obtained recoveries and matrix effects have typically not been stated or assessed. In the present study a solid-phase extraction (SPE) method was developed and validated for the simultaneous extraction of cholesterol and five key steroid hormones pregnenolone, 17-hydroxyprogesterone, testosterone, cortisol and aldosterone from H295R incubation medium, and finally detected by LC–MS/MS. Cholesterol was recovered at a level of 55.7%, while steroid hormone recoveries ranged from 98.2 to 109.4%. Matrix effects varied between −0.6% and 62.8%. Intra-day precision was deemed acceptable, but the inter-day precision for pregnenolone and aldosterone exceeded the precision limit of 15% RSD. Although LLE has been the most frequently used extraction method in H295R studies, this investigation has shown that SPE may relatively easily extract and recover steroid hormones, potentially replacing LLE. Section 2.3 teaches the sample preparation. The final SPE method contained the following procedural steps. In order to stabilize the steroid hormones, 1.5 mL H295R incubation medium was initially pH adjusted to pH 3.0 ± 0.1 with diluted sulfuric acid. Subsequently each sample was spiked with 20.0 µL of the IS mixture (described in Section 2.1). SPE was performed using C18 cartridges preconditioned with 2 × 3 mL heptane, 3 mL acetone, 2 × 3 mL methanol and lastly with 2 × 3 mL tap water adjusted to pH 3.0. Samples were quantitatively transferred to the cartridges by flushing the sample test tube twice with 1 mL of pH-adjusted tap water and extracted at a rate of 1–2 mL/min using a vacuum manifold. Immediately after enrichment, the SPE cartridges were washed with 2 × 3 mL tap water (pH 3.0) followed by air-drying using the vacuum manifold for 30 min. Finally, analytes were eluted from the SPE cartridges with 7 mL mobile phase B into a test tube and evaporated to dryness under a gentle stream of nitrogen. Evaporation was followed by reconstitution in 1.5 mL of a 50:50 mixture of mobile phases A and B, aided by 3 min of whirl mixing at 2600 rpm.
It would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Draisci solid-phase extraction method to include a was having a pH of 2 to 4 as taught by Abdel-Khalik because of the similarity in the solid-phase (C18), the recovery of analytes taught by Abdel-Khalik and the inclusion of testosterone in both methods.
Claims 8-9 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Karvaly in view of Schiffer as applied to claim 1 above, and further in view of He (Talanta 2014, newly cited and applied) and Pavlovic (Journal of Separation Science 2010, newly cited and applied). Karvaly teaches a Phenomenex StrataTM-X 33 µm, 60 mg, 2 mL polymeric reversed phase 96-well plates solid phase but does not teach that the SPE solid phase is magnetic particles or that the solid phase of the SPE is coated with a polymer matrix comprising at least one positively charged nitrogen atom.
In the paper He teaches a novel hydrophilic–lipophilic balanced magnetic nanoparticle, magnetic poly(divinylbenzene-co-N-vinylpyrrolidone) (HLB-MPNP) was successfully synthesized and applied for the extraction and determination of triazine and organochlorine pesticides in environmental water samples. The specific ratio of two monomers, hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene, endowed the magnetic nanoparticles with hydrophilic–lipophilic balanced character, which made it capable of extracting both polar and nonpolar analytes. The experimental parameters affecting extraction efficiency, including desorption conditions, sample pH, sample volume and extraction time were investigated and optimized. Under the optimum conditions, good linearity was obtained in the range of 0.20–10 μg L-1 for triazine herbicides and 5.0–100 ng L-1 for organochlorine pesticides, with correlation coefficients ranging from 0.994 to 0.999. The limits of determination were between 0.048 and 0.081 μg L-1 for triazine herbicides and 0.39 and 3.26 ng L-1 for organochlorine pesticides. The proposed method was successfully applied in the analysis of triazine and organochlorine pesticides in environmental water samples (ground, river and reservoir). The introduction on page 1 teaches that magnetic solid-phase extraction (MSPE) based on functionalized magnetic materials had received considerable attention in recent years, especially as a promising sample preparation technique. In MSPE, magnetic adsorbent is added to the solution for the adsorption of the analytes. The adsorbent with the adsorbed analyte is then separated from the solution using a magnet. The analyte is consequently eluted and analyzed. Compared with traditional SPE, the phase separation process in MSPE is easier and faster without the need of additional filtration procedure in SPE, because of the use of magnetic field. Adsorbent material is the most important component of the MSPE technique. Around that time, polymer coated magnetic nanoparticles (MNPs) had gained considerable attention. Polymer coating endows the MNPs with a diversity of adsorption selectivity. Besides, the polymer coating provides the MNPs with protection from aggregation and oxidization. But, for most of the polymer coated MNPs, polystyrene is main component. All the magnetic adsorbents mentioned above present an undiversified structure, either hydrophobic or hydrophilic. Their interactions with the analytes are basically hydrophobic and π–π interactions. For this reason, these magnetic adsorbents present low recoveries for the polar compounds or are too specific to a particular analyte. To overcome this drawback, a hydrophilic–lipophilic balanced magnetic material was needed, and polymer coated magnetic material is a good candidate. There are plenty of monomers of interest to choose. In this work, they introduced a hydrophilic monomer N-vinylpyrrolidone into the polymer preparation inspired by the Waters Oasis HLB SPE sorbent. A certain ratio of hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene made the polymer hydrophilic–lipophilic balanced. The prepared magnetic polymer nanoparticles present good extraction performance for both polar and non-polar compounds. Section 2.3.2 presents the preparation of the hydrophilic–lipophilic balanced magnetic polymer nanoparticles (HLB-MPNPs). Section 2.4 presents the extraction procedure using the magnetic particles. HLB-MPNPs (60 mg) were first activated by adding 100 μL of methanol, and then dispersed in 200 mL water samples by ultrasonic irradiation. After adsorption equilibrium (20 min for triazine herbicides; 5 min for OCPs), the magnetic adsorbent was isolated from the suspension with an Nd–Fe–B strong magnet. The supernatant was decanted and the residue solution and magnetic adsorbent were transferred to a 10 mL plastic centrifuge tube. The magnetic adsorbent was again aggregated by a magnet to remove the residue solution completely by a syringe. The analytes were eluted from the magnetic adsorbent with suitable solvent. Subsequently, the desorption solutions were mixed into a 5 mL centrifuge tube and evaporated to dryness at 30 °C under a stream of nitrogen. The residue was redissolved in 200 μL of solvent (methanol for triazine herbicides; n-hexane for OCPs) and filtered through a PTEE filter (0.22 μm) before chromatographic analysis. Section 3.5 compared the developed method to other extraction methods including SPE and micro SPE. The MSPE method's ability to extract both polar and nonpolar analytes was seen as the greatest advantage compared to other methods. Additionally, the presented method was comparable or superior to other methods in terms of LOD, enrichment factors (EFs), extraction time and organic solvent used (see Table 4).
The Pavlovic paper teaches the development and optimization of the SPE procedure for determination of pharmaceuticals in water samples by HPLC-diode array detection. Relative to the instant claims are table 2 showing different SPE cartridge materials and their surface modifications and Table 5 showing that both the OASIS HLB and Stata-X commercial materials use a pyrrolidone modification of the polymer.
It would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Karvaly process to use the polymer coated magnetic nanoparticles of He as the solid phase material because of the advantages taught by He for the magnetic solid phase particles compared to conventional SPE and/or micro SPE and the similarity in the Strata-X and OASIS HLB solid phase materials as shown by the teachings of Pavlovic.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. The additionally cited art is related to steroid analysis and solid phase extraction materials and methods. Of note with respect to the analysis of steroids is the cited Elmongy paper (Journal of Chromatography A 2020) having a disclosure that appears to anticipate one or more of the instant claims for reasons similar to those of the above applied anticipatory references. Relevant to the solid-phase extraction materials and process in the cited Liu paper (Science of the Total Environment 2020) teaching the preparation and use of magnetic solid-phase extraction with hydrophilic-lipophilic-balanced materials for LC-MS/MS analysis.
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/ARLEN SODERQUIST/ Primary Examiner, Art Unit 1797