DETAILED ACTION
The amendment filed on 12/01/2025 has been entered and fully considered. Claims 1-16 and 18 are pending, of which claim 1, 3-5 and 18 are amended.
Response to Amendment
In response to amendment, the examiner modifies rejection over the prior art established in the previous Office action.
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 .
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-12, 14-16 and 18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Osteresch et al. (Analytical Bioanalytical Chemistry, 2017, IDS) (Osteresch) in view of Devreese et al (Journal of Chromatography A, 2012) (Devreese).
Regarding claim 1, Osteresch taches a method for the detection of mycotoxins and metabolites in human (abstract),
Aflatoxin B1 (AFB1),
Aflatoxin M1 (AFM1),
Fumonisin (FB1),
Fumonisin (FB2),
Ochratoxin A (OTA),
Deoxynivalenol (DON),
T2 toxin (T2),
HT-2-toxin (HT2),
Zearalenone (ZEN), (Table 1),
The method comprising:
- collecting the blood of human as a dried blood sample (page 3371, par 2);
- preparing the dried blood sample for analysis (page 3371, par 3);
- analyzing the prepared dried blood sample by liquid chromatography-tandem
mass spectrometry (LC-MS/MS):
a) detecting ZEN (Table 1) in an LC-MS/MS step, including:
- the reversed phase LC process using as mobile phase, a mixture of acetic acid, acetonitrile and water, the proportion of the acetonitrile solution over the water solution gradually increasing during the process, and at the end diminishing to recondition the LC column (page 3371, par 4),
- the mass spectrometer operating in negative electro-spray ionization mode (-14500 V) (Table 1, page 3371, par 4); and
b) detecting AFB1, AFM1, FB1, FB2, OTA, DON, T2, and HT2 (Table 1) in another LC-MS/MS step, including:
after the reversed phase LC process,
- the mass spectrometer operating in positive electro-spray ionization mode as well (+5500 V) (Table 1, page 3371, par 4).
Osteresch does not specifically teach that the step b) includes
- the reversed phase LC process using as mobile phase, a mixture of ammonium formate, formic acid, water and methanol, the proportion of the methanol solution over the water solution gradually increasing during the process, and at the end diminishing to recondition the LC column.
However, Ion detection modes in mass spectrometry, such as positive and negative ion modes, are determined by the charge of the ions being detected. In positive ion mode, the detector is set up to detect cations (ions with a positive charge), while in negative ion mode, it detects anions (ions with a negative charge). This is because the polarity of the applied electric field in the mass analyzer is reversed to attract ions of the opposite charge. The function of LC is to separate analytes in retention time before the MS detection. Therefore, while LC conditions changes, the ions are still detected in a mode based on their charges. That is the negatively charged ions are detected in negative ion mode, the positively charged ions are detected in positive ion mode. Claim 1 distinct from Osteresch by adding additional LC separation step with a different mobile phase, which does not change the ion diction mode in mass analyzer.
Thus, the underlying objective technical problem is providing an additional extraction solvent (mobile phase in the reverse phase column) for separation of the mycotoxins and metabolites. Devreese teaches both acetic acid/acetonitrile/water and formic acid/methanol/water as the extraction solvent for the mycotoxins and metabolites (Table 1, page 75, par 11). Devreese teaches that “For multi-mycotoxin screening analysis, two gradient elution programs were performed depending on the MS/MS detection mode, i.e. positive or negative electrospray ionization (ESI). The flow rate was set at 300 µL/min. An overview of the gradient programs is given in Table 1.” (page 75, par 11). Ammonium formate is a counter ion buffer to the formic acid. In reversed-phase chromatography, the mobile phase's extraction power is indeed a crucial factor influencing separation. A mobile phase with a good extraction power can better dissolve and elute compounds from the stationary phase, leading to improved separation efficiency.
Thus, it would have been obvious to one of ordinary skill in the art to also try a mixture of ammonium formate, formic acid, water and methanol as the extraction solvent in the reverse phase column as taught by Devreese and gradually increasing the extraction (methanol) concentration during the process as taught by Devreese, in order to separate and detect more mycotoxins and metabolites, because Devreese demonstrated that analyzing the blood sample of a pig by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in two gradient programs can detect more mycotoxins and metabolites in the sample. The result is predictable.
Osteresch does not teach that the dried blood sample is from broiler chickens or pigs. However, a person skilled in the art would have recognized that Osteresch’s method can readily be used for analyzing dried blood sample from broiler chickens or pigs for detection of one or more mycotoxin(s) and metabolites. Devreese teaches that the blood sample can be from pigs for detection of one or more mycotoxin(s) and metabolites (page 75, par 6-7).
Regarding claim 2, Osteresch teaches that the method is for the further detection of the following mycotoxins and metabolites:
Alternariol methyl ether (AME),
Alternariol (AOH),
Tenuazonic Acid (TEA),
Beauvericin (BEA).,
Enniatin A, A1, B, B1 (ENNA, ENNA1, ENNB, ENNB1)
(Table 1).
Regarding claim 3, Osteresch teaches that the method is for the further detection of the following mycotoxins and metabolites:
de-epoxy-deoxynivalenol (DOM1),
15-acetyldeoxynivalenol (15ADONJ,
3-acetyldeoxynivalenol (3ADON),
Zearalanone (ZAN),
a-Zearalenol (AZEL),
a-Zearalanol (AZAL),
p-Zearalanol (BZAL),
p-Zearalenol (BZEL),
DON-glucuronide (DON-GlcA),
DON-Sulphate (DON-S),
ZEN-glucuronide (ZEN-GlcA),
a-zearaienol-glucuronlde (A-ZEL-GlcA),
P-zearalenol-glucuronide (B-ZEL-GkA),
(Table 1).
Regarding claim 4, Osteresch teaches that wherein any of the following mycotoxins and met.abolites are detected by the mass spectrometer operating in negative electrospray ionization mode:
zearalanone (ZAN),
n-zearalenol (AZEL)
n-zearalanol (AZAL),
P-zeara lan ol (BZAL),
P-zeara lenol (BZEL),
tenuazonic acld (TEA),
alternarlol (AOH).
alternariol methyl ether (AME).
deoxynlvaienol sulphate (DON-SJ)
deoxynlvaienol glucuronide (DON-GlcA),
zearalenone glucuronide (ZEN-GlcA),
a-ZEL-glucuronide (AZEL-GlcA),
b-ZEL- giucuronide (BZEL-GlcA)
(Table 1).
Regarding claim 5, Osteresch teaches that wherein any of the following mycotoxins and metabolites are detected by the mass spectrometer operating in positive electrospray ionization mode:
de-epoxy-deoxynivalenol (DOM1),
3-acetyldeoxynivalenol (3ADON),
15-acetyldeoxynivalenol (15ADON),
enniatin A1 (ENNA1),
enniatin A (ENNA),
enniatin B (ENNB),
enniatin B1(ENNB1),
beauvericin (BEA)
(Table 1).
Regarding claim 6 and 16, It would have been obvious to one of ordinary skill in the art to optimize the timing and concentration of the mobile phase of the reverse column by routine experimentation.
Regarding claim 7, Osteresch teaches that wherein ln the LC process a column is used comprising an hydrophobic stationary phase comprising silica with covalently bonded alkyl chains, preferably high strength silica with trifunctional C18 alkyl bonded chains (page 3371, par 4).
Regarding claim 8, Osteresch teaches that the method comprising, prior to analyzing, extracting the mycotoxins and the metabolites from the dried blood sample in an extraction solvent and subjecting the extraction solvent and the dried blood sample to an ultrasonic bath treatment (page 3371, par 3).
Regarding claim 9, Osteresch teaches that whereby the extraction solvent comprises a water /acetonitrile /acetone mixture (page 3371, par 4).
Regarding claim 10, Osteresch teaches that the method comprising drying the extraction solvent and reconstituting the dried mass in a reconstitution solvent (page 3371, par 3).
Regarding claim 11, Devreese teaches that whereby the reconstituting solvent comprises a water/methanol/formic acid mixture (Table 1).
Regarding claim 12, Osteresch teaches that the method further comprising spiking the collected dried blood sample with one or more internal standards (Fig. 1, page 3373, par 4).
Regarding claim 14-15, Osteresch teaches the method for the assessment of the exposure of humans and animals to feed contaminated with mycotoxins (page 3370, par 3). Thus, it would have been obvious to one of ordinary skill in the art to use the same method as a biomonitoring tool for the assessment of the exposure of farming production pigs or broiler chickens to feed contaminated with mycotoxins, or for assessing the impact of the addition of mycotoxin detoxifying agents to animal feed.
Devreese also teaches use of the method as a biomonitoring tool for screening and assessing the exposure of farming production pigs or broiler chickens to feed contaminated with mycotoxins (abstract).
Regarding claim 18, Osteresch teaches detecting the following mycotoxins and metabolites in the LC-MS/MS step in which the mass spectrometer is operated in the negative electrospray ionization mode:
o zearalanone (ZAN),
o a-zearalenol (AZEL)
o a-zearalanol (AZAL),
o ~-zearalanol (BZAL),
o ~-zearalenol (BZEL),
o tenuazonic acid (TEA),
o alternariol (AOH),
o alternariol methyl ether (AME),
o deoxynivalenol sulphate (DON-S),
o deoxynivalenol glucuronide (DON-GlcA),
o zearalenone glucuronide (ZEN-GlcA),
o a-ZEL-glucuronide (AZEL-GlcA),
o b-ZEL- glucuronide (BZEL-GlcA)
(Table 1); and
detecting the following mycotoxins and metabolites in the LC-MS/MS step in which the mass spectrometer is operated in the positive electrospray ionization mode:
o de-epoxy-deoxynivalenol (DOMl),
o 3-acetyldeoxynivalenol (3ADON),
o 15-acetyldeoxynivalenol (15ADON),
o enniatin A1 (ENNA1),
o enniatin A (ENNA),
o enniatin B (ENNB ),
o enniatin B1(ENNB1),
o beauvericin (BEA)
(Table 1).
Claim 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Osteresch in view of Devreese as applied to claims 1-12 and 14-18 above, and further in view of Leblanc et al. (US 2019/0187151, IDS) (Leblanc).
Regarding claim 13, Osteresch does not specifically teach to isotopic label the mycotoxin and metabolites as internal standard. Leblanc teaches spiking the sample with one or more isotope labeled biomarker as the internal standards (abstract). It would have been obvious to one of ordinary skill in the art to isotopic label the mycotoxin and metabolites as internal standard, in order to quantify the mycotoxin and metabolites in a sample.
Response to Arguments
Applicant’s arguments with respect to claim(s) 1 have been considered but are moot in view of new ground of rejection.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to XIAOYUN R XU, Ph. D. whose telephone number is (571)270-5560. The examiner can normally be reached M-F 8am-5pm.
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797
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