Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Arguments
Applicant’s amendments, see pg 9, filed 07/23/2026, with respect to claim 10 have been fully considered. The objection of 04/24/2026 has been withdrawn.
Applicant’s arguments, see 9-11, filed 07/23/2026, with respect to the rejection(s) of claims 1-16 under 35 USC § 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made, still in part relying on the previously applied prior art including Jenkinson, Zeng, Wu, and Li.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-16 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
Step 1: Claim 1 recites the steps for simultaneously detecting 11 vitamin D by liquid chromatography-mass spectrometry. Thus, the claim is a method, which is one the statutory categories of invention. Claim 2-16 also are dependent on Claim 1.
Step 2A Prong One: Claim 1 recite a judicial exception and identify the abstract idea/law of nature/natural phenomenon. Claim 1 recites “preparing a standard curve equation” and “substituting a detection result of the incoming sample into the standard curve equation”. This claim recites preparing and plugging in data to an equation which is mathematical concept. The mathematical concepts grouping is defined as mathematical relationships, mathematical formulas or equations, and mathematical calculations (See MPEP 2106.04(a)(2)). Thus, the steps of preparing a standard curve equation and plugging in data to the standard curve would fall under the abstract idea groups of mathematical concepts. (Step 2A -Prong 1: Yes)
Step 2A Prong Two: The judicial exception is not integrated into a practical application because the claims do not impose any meaningful limits on practicing the abstract idea. Claim 1 further reciting sample preparation steps claimed at a high level of generality and using LC-MS/MS which are data gathering steps which are considered extra solution activities (See MPEP 2106.05 (g)). Accordingly, this additional element does not integrate the abstract idea into a practical application because it does not impose any meaningful limits on practicing the abstract idea. The claim is directed to an abstract idea. (Step 2A -Prong 2: No)
Step 2B: Claims 1 does not have any steps or features which are significantly more and the steps are well known in the art as taught in the prior art below. Claim 1 further reciting sample preparation steps claimed at a high level of generality and detection by LC-MS/MS which is well understood and routine and conventional. Claim 1 is ineligible. Claims 2-16 do not appear to have ‘significantly’ more. Claims 2-16 recite sample preparation steps such as “centrifuging”, vortexing”, “derivatization”, as well as using LC-MS/MS, claimed at a high level of generality which are well understood routine and conventional as referenced in the prior art below. Since it is claimed at a high level of generality, there are no meaningful limitation claimed, such as a particular or unconventional machine or transformation of a particular article. (Step 2B: No)
Thus, claim 1 is ineligible. Claims 2-16 are dependent on Claim 1, and are also rejected.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claims 1, 2, 3, 5, 7, 8, 9, 10, 14, 15, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over journal article "Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry" by Jenkinson et al in view of Holmquist et al. (US 20160061848 A1) and Yu et al (CN 108593790 B). The examiner has obtained a machine translation of the CN document above from Espacenet. The rejection below is based off the machine translation.
Regarding Claim 1, Jenkinson teaches preparing a standard curve equation, comprising of preparing an internal standard working liquid and a standard working liquid, preparing a sample solution for standard curve, and detecting the sample solution for standard curve by using the LC-MS/MS to obtain the standard curve equation for calculating contents of the vitamins D in blood (pg. 1644, left column).
Furthermore, Jenkinson teaches pre-treating a sample to be detected, comprising of uniformly mixing the internal standard working liquid, the sample to be detected, and a protein precipitant to obtain a first resulting mixture, extracting the first resulting mixture twice with an extractant to obtain a total supernatant, adding a derivatization reagent to a part of the total supernatant to obtain a second resulting mixture, uniformly mixing the second resulting mixture for a derivatization treatment to obtain a third resulting mixture, blow-drying the third resulting mixture, and adding a remaining part of the total supernatant to the third resulting mixture to obtain a fourth resulting mixture, blow-drying the fourth resulting mixture again, adding a reconstitution solution to the fourth resulting mixture to obtain a fifth resulting mixture, uniformly mixing the fifth resulting mixture, and centrifuging the fifth resulting mixture and taking a supernatant of the fifth resulting mixture as an incoming sample; the extractant is a mixed solution of normal hexane (pg. 1644, right column).
Furthermore, Jenkinson teaches detecting the incoming sample, comprising of detecting the incoming sample by using the LC-MS/MS, and substituting a detection result of the incoming sample into the standard curve equation to obtain the contents of the vitamins D in the sample to be detected (pg. 1644, right column).
Jenkinson does not teach the specific 11 vitamin D which are D2, D3, 1,25-(OH)2D2, 1,25-(OH)2D3, 25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D2, 3-epi-25-(OH)D3, 24,25-(OH)2D2, 24,25-(OH)2D3, and 3-epi-24,25-(OH)2D3 as required by claim 1 mentioned above. Jenkinson teaches the following vitamin D which are 25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, 3-epi-25(OH)D2, 24,25(OH)2D3, 1,25(OH)2D3, 1,25(OH)2D2, 25(OH)D3-d3, 25(OH)D2-d3, 3-epi-25(OH)D2-d3, 24,25(OH)2D3-d6 and 1,25(OH)2D3-d3, (pg. 1644, left column).
Furthermore, Jenkinson does not teach D2, D3, 24,25-(OH)2D2, and 3-epi-24,25-(OH)2D3 and does not teach the pretreatment requires mixing an underivatized fraction of the extracted sample with a derivatized fraction of the extracted sample.
Holmquist teaches quantitative measurements of vitamin D compounds by mass spectrometry (Abstract). Holmquist further teaches the vitamin D compounds selected consisting of vitamin D2 and vitamin D3 (para 0012). Holmquist further teaches processing each test sample differently to form a plurality of processed samples and combining the processed samples to form a multiplex sample (para 0009). Holmquist further teaches processing a test sample comprises subjecting each test sample to a different derivatizing agent and processing without subjecting the sample to a derivatizing agent (para 0010). Thus, it would be obvious to one of ordinary skill in the art before the effective filing date to modify Jenkinson with vitamin D2 and D3 and the pretreatment requires mixing an underivatized fraction of the extracted sample with a derivatized fraction of the extracted sample as taught by Holmquist for the benefit of distinguishing compounds by mass spectrometry that were not originally distinguishable (para 0109).
Jenkinson in view of Holmquist does not teach separating and determining 24,25-(OH)2D2, and 3-epi-24,25-(OH)2D3.
However, Yu teaches a method for the simultaneous detection of serum 24,25(OH)2D and 25OHD by liquid chromatography-tandem mass spectrometry (pg 4, para 0003). Yu further teaches finding 24,25-(OH)2D2 (pg 4, para 0006), and 3-epi-24,25-(OH)2D3 (pg 5, para 0008) for the benefit of determining which metabolite is present in the patient sample for clinical purposes (pg 4, para 0006). Thus, it would be obvious to one of ordinary skill in the art before the effective filing date to modify Jenkinson in view of Holmquist with separating and determining 24,25-(OH)2D2, and 3-epi-24,25-(OH)2D3 as taught by Yu for the benefit of determining which metabolite is present in the patient sample for clinical purposes (pg 4, para 0006).
Regarding Claim 2, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 1. Jenkinson further teaches preparing a first internal standard working liquid, a second internal standard working liquid, a first standard working liquid, and a second standard working liquid (pg. 1644, left column). Jenkinson teaches the preparation of standard solutions with 25(OH)D3-d3, 25(OH)D2-d3, 3-epi-25(OH)D3-d3, 1,25(OH)2D3-d3, 24,25(OH)2D3-d6 and 20OHD3-d3 (pg. 1644, left column).
Jenkinson does not teach the first internal standard working liquid containing isotopic internal standards of D2, D3, 1,25-(OH)2D2, and 1,25-(OH)2D3, the second internal standard working liquid containing isotopic internal standards of 25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D2, 3-epi-25-(OH)D3, 24,25-(OH)2D2, and 24,25-(OH)2D3, the first standard working liquid containing standard solutions of D2, D3, 1,25-(OH)2D2, and 1,25(OH)2D3, and the second standard working liquid containing standard solutions of 25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D2, 3-epi-25-(OH)D3, 24,25-(OH)2D2, 24,25-(OH)2D3, and 3-epi-24,25-(OH)2D3.
Although, Jenkinson does not teach the specific vitamin D internal standards as required by claim 2 mentioned above, it does teach the preparation of a standard solutions (pg. 1644, left column). The standard solutions can be adjusted to include various types of Vitamin D to accurately measure simultaneously vitamin D to gain a greater understanding of vitamin D metabolism and its role in health and disease. Jenkinson only teaches some the vitamin D metabolites, however, the vitamin D metabolites that are not mentioned are structurally similar and with similar utility. It can be reasonable expected that the structural similarity will have similar properties (pg. 1642-1643). See MPEP 2144.09(I). Thus, it would be obvious to a person of ordinary skill in the art to modify the standard solutions of Jenkinson with the solutions in claim 2 to accurately measure simultaneously vitamin D to gain a greater understanding of vitamin D metabolism and its role in health and disease.
Further regarding Claim 2, Jenkinson teaches mixing the first standard working liquid and the first internal standard working liquid to prepare a standard curve working liquid with a gradient concentration (Table 2) and blow-drying the standard curve working liquid, adding the derivatization reagent to the standard curve working liquid for the derivatization treatment to obtain a derived standard curve working liquid, blow-drying the derived standard curve working liquid and respectively adding the second standard working liquid and the second internal standard working liquid with corresponding concentrations according to a concentration gradient to obtain a sixth resulting mixture, adding the reconstitution solution to the sixth resulting mixture to obtain a seventh resulting mixture, and mixing the seventh resulting mixture to obtain the sample solution for standard curve (pg. 1644, right column); and detecting the sample solution for standard curve by using the LC-MS/MS to obtain the standard curve equation (pg. 1644, right column).
Regarding Claim 3, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 2. Jenkinson further teaches preparing the standard working solutions by diluting the various types of vitamin D internal standards in methanol (pg. 1644, left column).
Jenkinson does not teach the standard working solutions by diluting the various types of vitamin D internal standards in 70%-100% methanol.
Although Jenkinson reference does not include a range of percent by volume for methanol, it is not inventive to discover the optimum range of percent by volume. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be with the level of skill of the one of ordinary skill in the art to determine a suitable volume range of methanol to dilute the internal standards to minimize the matrix effect.
Regarding Claim 5, Jenkinson in view of Holmquist and further in view of Yu suggests the claimed invention. Jenkinson further teaches the preparation of internal standard to standard working solutions (pg. 1644, left column) as well as preparing the derivatization reagent (pg. 1644, right column) and the reconstitution solution (pg. 1644, right column). Claim 5 filed on 07/23/2026 recited "the method according to claim 2 to 3" which should recite "the method according to claim 2" as stated the claim set filed on 10/20/2023. For examination purposes, claim 5 has been interpreted as being dependent on claim 2.
Jenkinson does not teach a volume ratio of the first standard working liquid to the first internal standard working liquid is 1:1-2:1; a volume ratio of the first internal standard working liquid to the derivatization reagent is 1:10-1:20; a volume ratio of the first standard working liquid to the second standard working liquid is 1: 1; and a volume ratio of the second standard working liquid to the second internal standard working liquid II and the reconstitution solution is 2:1:7.
Although Jenkinson reference does not include various ratios of standard working solutions to other solutions and reagents, it is not inventive to discover the optimum ratio for the various solutions. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be obvious to one of ordinary skill in the art to determine a suitable ratio of the first standard working liquid to the first internal standard working liquid for the benefit of accuracy and precision in detection. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the first internal standard working liquid to the derivatization reagent for the benefit of achieving sensitivity for measuring certain vitamin D. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the second standard working liquid to the second internal standard working liquid II and the reconstitution solution for the benefit of accuracy and precision and minimizing the matrix effect. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the first standard working liquid to the second standard working liquid for the benefit of normalizing the data.
Regarding Claim 7, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 2. Jenkinson further teaches the preparation of internal standard to standard working solutions (pg. 1644, left column), preparing the protein precipitant (pg. 1644, right column), collecting the supernatant (pg. 1644, right column), and preparing the reconstitution solution (pg. 1644, right column). Jenkinson also teaches mixing the working standards solutions with the protein precipitant (pg. 1644, right column).
Jenkinson does not teach volume ratio of the first internal standard working liquid to the second internal standard working liquid is 1:1, a volume ratio of the sample to be detected to the protein precipitant is 1:1-2:1; a volume ratio of the part of the total supernatant for the derivatization treatment to the remaining part of the total supernatant is 1:1; and a volume ratio of the reconstitution solution to the sample to be detected is 1:1-1:2.
Although Jenkinson reference does not include various ratios as mentioned in claim 7 of the instant invention, it is not inventive to discover the optimum ratio for the various solutions. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be obvious to one of ordinary skill in the art to determine a suitable ratios of sample to protein precipitant for the benefit of maximizing the protein dissociation to improve accuracy of detecting vitamin D. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of first internal standard working liquid to the second internal standard working liquid for the benefit of normalizing the data. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of sample to reconstitution solution for the benefit of minimizing the matrix effect. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the part of the total supernatant for the derivatization treatment to the remaining part of the total supernatant for the benefit of reducing excess waste in the experiment.
Regarding Claim 8, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 7. Jenkinson further teaches carrying out a vortex mixing the first internal standard working liquid (pg. 1644, right column), the second internal standard working liquid, the sample to be detected and the protein precipitant to obtain the first resulting mixture (pg. 1644, right column), extracting the first resulting mixture twice with the extractant to obtain the total supernatant, adding the derivatization reagent to the part of the total supernatant to obtain the second resulting mixture (pg. 1644, right column), carrying out the derivatization treatment on the second resulting mixture after the vortex to obtain the third resulting mixture, blow-drying the third resulting mixture, adding the remaining part of the total supernatant to the third resulting mixture to obtain the fourth resulting mixture (pg. 1644, right column), blow-drying again the fourth resulting mixture, adding the reconstitution solution to the fourth resulting mixture to obtain the fifth resulting mixture, carrying out the vortex mixing on the fifth resulting mixture, centrifuging the fifth resulting mixture at a rotating speed of, and taking the supernatant of the fifth resulting mixture as the incoming sample (pg. 1644, right column).
Jenkinson does not teach carrying out a vortex mixing the first internal standard working liquid, the second internal standard working liquid, the sample to be detected and the protein precipitant at a rotating speed of 1,500-2,500 rpm for 3-5 min to obtain the first resulting mixture, carrying out the derivatization treatment on the second resulting mixture after the vortex mixing at the rotating speed of 1,500-2,500 rpm for 30 s to 1 min to obtain the third resulting mixture, and carrying out the vortex mixing at the rotating speed of 1,500-2,500 rpm for 1-3 min on the fifth resulting mixture, centrifuging the fifth resulting mixture at a rotating speed of 12,000-14,000 rpm for 5-10 min, and taking the supernatant of the fifth resulting mixture as the incoming sample. Although Jenkinson reference does not include the rotating speed of vortex mixing the solution, it is not inventive to determine the optimum rotating speed of mixing the solutions throughout the procedure. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be with the level of skill of the one of ordinary skill in the art to determine the optimum rotating speed of mixing the solutions throughout the procedure for the benefit of ensuring the solutions have been mixed thoroughly.
Regarding Claim 9, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 1. Jenkinson further teaches the extraction procedure (pg. 1644, right column).
Jenkinson does not teach extracting twice with the extractant comprises: during a first extraction, a volume ratio of the sample to be detected to the extractant is 2:6.5-1:5; and a volume ratio of the extractant used during a second extraction to the extractant used during the first extraction is 0.8-1:1.
Although Jenkinson reference does not include a ratio of extractant to sample and a ratio between the extractant used both times, it is not inventive to discover the optimum ratio for the amount of extractant to the sample. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be with the level of skill of the one of ordinary skill in the art to determine the optimum ratio of the extractant to the sample to maximize the sample recovered as well as the ratio of the extractant used during a second extraction to the extractant used during the first extraction for the benefit of maximizing the amount of sample recovered efficiently.
Regarding Claim 10, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 9. Jenkinson further teaches wherein during the first extraction, the internal standard working liquid, the sample to be detected, and the protein precipitant are mixed uniformly to obtain the first resulting mixture (pg. 1644, right column), and then the extractant is added to the first resulting mixture to obtain a sixth resulting mixture, and the sixth resulting mixture is subjected to a vortex mixing, and centrifuged, and a supernatant of the sixth resulting mixture is taken as a first supernatant (pg. 1644, right column); the extractant is added into a centrifuged precipitate of the sixth resulting mixture to obtain a seventh resulting mixture, subjected to the vortex mixing, a supernatant of the seventh resulting mixture is taken as a second supernatant, and the first supernatant and the second supernatant are combined to obtain the total supernatant (pg. 1644, right column).
Jenkinson does not teach the sixth resulting mixture is subjected to a vortex mixing at a rotating speed of 1,500-25,00 rpm for 3-5min, and centrifuged at a rotating speed of 12,000-14,000 rpm for 5-10 min, and a supernatant of the sixth resulting mixture is taken as a first supernatant; the extractant is added into a centrifuged precipitate of the sixth resulting mixture to obtain a seventh resulting mixture, subjected to the vortex mixing at the rotating speed of 1,500-2,500 rpm for 3-5min, centrifuged at the rotating speed of 12,000-14,000 rpm for 5-10 min.
Although Jenkinson reference does not include the rotating speed of mixing the solution, it is not inventive to discover the optimum rotating speed of solutions throughout the procedure. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be with the level of skill of the one of ordinary skill in the art to determine the optimum rotating speed of mixing the solutions throughout the procedure to optimize the procedure for the benefit of ensuring the solutions have been mixed thoroughly.
Regarding Claim 14, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 3. Jenkinson further teaches the preparation of internal standard to standard working solutions (pg. 1644, left column) as well as preparing the derivatization reagent (pg. 1644, right column) and the reconstitution solution (pg. 1644, right column).
Jenkinson does not teach a volume ratio of the first standard working liquid to the first internal standard working liquid is 1:1-2:1; a volume ratio of the first internal standard working liquid to the derivatization reagent is 1:10-1:20; a volume ratio of the first standard working liquid to the second standard working liquid is 1: 1; and a volume ratio of the second standard working liquid to the second internal standard working liquid II and the reconstitution solution is 2:1:7.
Although Jenkinson reference does not include various ratios of standard working solutions to other solutions and reagents, it is not inventive to discover the optimum ratio for the various solutions. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be obvious to one of ordinary skill in the art to determine a suitable ratio of the first standard working liquid to the first internal standard working liquid for the benefit of accuracy and precision in detection. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the first internal standard working liquid to the derivatization reagent for the benefit of achieving sensitivity for measuring certain vitamin D. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the second standard working liquid to the second internal standard working liquid II and the reconstitution solution for the benefit of accuracy and precision and minimizing the matrix effect. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the first standard working liquid to the second standard working liquid for the benefit of normalizing the data.
Regarding Claim 15, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 3. Jenkinson further teaches the preparation of internal standard to standard working solutions (pg. 1644, left column), preparing the protein precipitant (pg. 1644, right column), collecting the supernatant (pg. 1644, right column), and preparing the reconstitution solution (pg. 1644, right column). Jenkinson also teaches mixing the working standards solutions with the protein precipitant (pg. 1644, right column).
Jenkinson does not teach volume ratio of the first internal standard working liquid to the second internal standard working liquid is 1:1, a volume ratio of the sample to be detected to the protein precipitant is 1:1-2:1; a volume ratio of the part of the total supernatant for the derivatization treatment to the remaining part of the total supernatant is 1:1; and a volume ratio of the reconstitution solution to the sample to be detected is 1:1-1:2.
Although Jenkinson reference does not include various ratios as mentioned in claim 15 of the instant invention, it is not inventive to discover the optimum ratio for the various solutions. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be obvious to one of ordinary skill in the art to determine a suitable ratios of sample to protein precipitant for the benefit of maximizing the protein dissociation to improve accuracy of detecting vitamin D. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of first internal standard working liquid to the second internal standard working liquid for the benefit of normalizing the data. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of sample to reconstitution solution for the benefit of minimizing the matrix effect. It would be obvious to one of ordinary skill in the art to determine a suitable ratios of the part of the total supernatant for the derivatization treatment to the remaining part of the total supernatant for the benefit of reducing excess waste in the experiment.
Regarding Claim 16, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 8. Jenkinson further teaches the extraction procedure (pg. 1644, right column).
Jenkinson does not teach extracting twice with the extractant comprises: during a first extraction, a volume ratio of the sample to be detected to the extractant is 2:6.5-1:5; and a volume ratio of the extractant used during a second extraction to the extractant used during the first extraction is 0.8-1:1.
Although Jenkinson reference does not include a ratio of extractant to sample and a ratio between the extractant used both times, it is not inventive to discover the optimum ratio for the amount of extractant to the sample. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be with the level of skill of the one of ordinary skill in the art to determine the optimum ratio of the extractant to the sample to maximize the sample recovered as well as the ratio of the extractant used during a second extraction to the extractant used during the first extraction for the benefit of maximizing the amount of sample recovered efficiently.
Claims 11 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over journal article "Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry" by Jenkinson et al. in view of Holmquist et al. (US 20160061848 A1) and Yu et al (CN 108593790 B) as applied to claim 1 above, and further in view of Zeng et al. (CN113640428 A). The examiner has obtained a machine translation of the CN document above from Espacenet. The rejection below is based off the machine translation.
Regarding Claim 11, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 1. Jenkinson further teaches the LC-MS/MS is used for a detection under the following high-performance liquid phase conditions: a pentafluorophenyl chromatographic column is adopted as a chromatographic column (pg. 1644, right column); mobile phases: a phase A is an aqueous solution containing 0.05%-0.2% formic; and a phase B is a methanol solution containing 0.05%-0.2% formic acid (pg. 1644, left column) a flow rate: 0.25-0.35 mL/min (pg. 1644, right column) and a gradient elution conditions (pg. 1644, right column).
Jenkinson does not teach ammonium formate in the mobile phase.
However, Zeng teaches the use of ammonium formate with formic acid in the mobile phase for the detecting 25-hydroxyvitamin D in blood for the benefit of higher peaks. Thus, it would be obvious to one of ordinary skill in the art to modify Jenkinson with adding ammonium formate taught by Zeng for the benefit of higher peaks in the chromatograph.
Jenkinson also does not teach a column temperature: 25-35°C, a sample volume: 10-20 L, and an analysis time: 8min; and the exact gradient elution scheme.
Although Jenkinson reference does not include certain LC-MS/MS parameters, it is not inventive to discover the optimum detector parameters to detect vitamin D in LC-MS/MS. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be with the level of skill of the one of ordinary skill in the art to determine a suitable detector parameters for the most ideal LC-MS/MS detection.
Regarding Claim 12, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 1. Jenkinson further teaches how to use LC-MS/MS in the detection of vitamin D metabolites.
Jenkinson does not teach detection under the following mass spectrum conditions: using an electrospray ion source (ESI) and a positive ion mode for a multi-reaction monitoring under an ion spray voltage: 5,000 V-5,500 V; an ion source temperature: 300-400°C; an atomizing gas: 45-55 psi; an auxiliary gas: 25-35 psi; a curtain gas: 20-25 psi; and a collision gas: 8-10 psi as well as ion pairs.
However, Zeng teaches detection under the following mass spectrum conditions: using an electrospray ion source (ESI) and a positive ion mode for a multi-reaction monitoring under an ion spray voltage: 5,000 V-5,500 V; an ion source temperature: 300-400°C; an atomizing gas: 45-55 psi; an auxiliary gas: 25-35 psi; a curtain gas: 20-25 psi; and a collision gas: 8-10 psi as well as ion pairs (n0032) for the benefit of improving the mass spectrometry detection sensitivity. Thus, it would be obvious to modify the teaching of Jenkinson with the ESI source taught by Zeng for the benefit of improving the mass spectrometry detection sensitivity.
Claims 6 is rejected under 35 U.S.C. 103 as being unpatentable over journal article "Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry" by Jenkinson et al. in view of Holmquist et al. (US 20160061848 A1) and Yu et al (CN 108593790 B) as applied to claim 5 above, and further in view of Wu et al. (CN115078559 A). The examiner has obtained a machine translation of the CN document above from Espacenet. The rejection below is based off the machine translation.
Regarding Claim 6, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 5. Jenkinson further teaches the vortex mixing the sample and adding derivatization reagent, and then adding the reconstitution solution (pg. 1644, right column).
Jenkinson does not teach after adding the reconstitution solution, the vortex mixing is carried out at a rotating speed of 1,500-2,500 rpm for 1-3 min to obtain the sample solution for standard curve.
However, Wu teaches a method for detecting of vitamin D, specifically 25-OH-D, using liquid chromatography and single quadropoly mass spectrometry. Wu teaches adding the reconstitution solution (para n0083), the vortex mixing is carried out for 5 mins (para n0083) to obtain the thoroughly mixed sample solution for standard curve. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05(II)(A). Thus, it would be obvious to a person of ordinary skill in the art to modify the method in Jenkinson with the vortex mixing the solution after adding reconstitution solution as taught by Wu for the benefit of thoroughly mixing the solutions.
Claims 4 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over journal article "Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry" by Jenkinson et al. in view of Holmquist et al. (US 20160061848 A1) and Yu et al (CN 108593790 B) as applied to claims 1 and 2 above, and further in view of Zeng et al. (CN113640428 A), Wu et al. (CN115078559 A), and Li et al. (CN115112810 A). The examiner has obtained a machine translation of the CN document above from Espacenet. The rejection below is based off the machine translation.
Regarding Claim 4, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 1. Jenkinson further teaches the derivatization reagent being PTAD, reconstitution solution, protein precipitant, and extractant (pg. 1644, right column).
Jenkinson does not teach the derivatization treatment lasting 60min, the reconstitution solution being made of an aqueous solution of formic acid and methanol, the protein precipitant being methanol and the extractant being n-hexane mixed with methyl tertbutyl ether with a volume ratio of 2:1-4:1.
However, Zeng teaches the derivatization treatment lasting for 60 mins (para n0012), and the protein precipitant being made of methanol (n0026) for the benefit of isolating the vitamin D in the sample solution for analysis. Thus, it would be obvious to one of ordinary skill in the art to modify the teachings of Jenkinson with the derivatization treatment lasting for 60 mins and the protein precipitant being made of methanol taught by Zeng for the benefit of isolating the vitamin D in the sample solution for analysis.
Furthermore, Wu teaches the reconstitution solution being made of an aqueous solution of 0.1% formic acid and 75% methanol (para n0083) for the benefit of improving mass spectrometry. Thus, it would be obvious to one of ordinary skill in the art to modify the teachings of Jenkinson and Zeng with the reconstitution solution being made of an aqueous solution of formic acid and methanol as taught by Wu for the benefit of improving mass spectrometry.
Furthermore, Li teaches sample pretreatment method for 25-hydroyvitamin D to be detected in HPLC-MS/MS. Li teaches the extractant being n-hexane mixed with methyl tertbutyl ether with a volume ratio of 3:1 (para n0024) for the benefit of improving extraction efficiency of the extractant. Thus, it would be obvious to one of ordinary skill in the art to modify the teachings of Jenkinson, Zeng and Wu with the extractant being n-hexane mixed with methyl tertbutyl ether with a volume ratio of 3:1 as taught by Li for the benefit of improving extraction efficiency of the extractant.
Regarding Claim 13, Jenkinson in view of Holmquist and further in view of Yu teaches the invention of claim 2. Jenkinson further teaches the derivatization reagent being PTAD, reconstitution solution, protein precipitant, and extractant (pg. 1644, right column).
Jenkinson does not teach the derivatization treatment lasting 60min, the reconstitution solution being made of an aqueous solution of formic acid and methanol, the protein precipitant being methanol and the extractant being n-hexane mixed with methyl tertbutyl ether with a volume ratio of 2:1-4:1.
However, Zeng teaches the derivatization treatment lasting for 60 mins (para n0012), and the protein precipitant being made of methanol (n0026) for the benefit of isolating the vitamin D in the sample solution for analysis. Thus, it would be obvious to one of ordinary skill in the art to modify the teachings of Jenkinson with the derivatization treatment lasting for 60 mins and the protein precipitant being made of methanol taught by Zeng for the benefit of isolating the vitamin D in the sample solution for analysis.
Furthermore, Wu teaches the reconstitution solution being made of an aqueous solution of 0.1% formic acid and 75% methanol (para n0083) for the benefit of minimizing the matrix effect. Thus, it would be obvious to one of ordinary skill in the art to modify the teachings of Jenkinson and Zeng with the reconstitution solution being made of an aqueous solution of formic acid and methanol as taught by Wu for the benefit of minimizing the matrix effect.
Furthermore, Li teaches the extractant being n-hexane mixed with methyl tertbutyl ether with a volume ratio of 3:1 (para n0024) for the benefit of improving extraction efficiency of the extractant. Thus, it would be obvious to one of ordinary skill in the art to modify the teachings of Jenkinson, Zeng and Wu with the extractant being n-hexane mixed with methyl tertbutyl ether with a volume ratio of 3:1 as taught by Li for the benefit of improving extraction efficiency of the extractant.
Conclusion
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/H.R.B./Examiner, Art Unit 1798
/CHARLES CAPOZZI/Supervisory Patent Examiner, Art Unit 1798
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