Identification of Penicillin G Metabolites under Various Environmental Conditions using LC-MS/MS

Transcription

1 Identification of Penicillin G Metabolites under Various Environmental Conditions using LC-MS/MS Acid Base Fadi Aldeek, Ph.D. Environmental Specialist III Fadi.Aldeek@FreshFromFlorida.com Florida Department of Agriculture and Consumer Services 53rd NACRW Annual Meeting, St. Pete Beach, FL, July 17-20, 2016

2 Citrus Greening Disease (Huanglongbing, HLB) Huanglongbing (HLB), also known as citrus greening disease, is caused by bacteria in the genus Candidatus Liberibacter. It is transmitted through an insect called the Asian citrus psyllid, which feeds on citrus foliage. This disease has emerged as one of the biggest threats to citrus trees worldwide. According to the United States Department of Agriculture s (USDA) National Agricultural Statistics Service (NASS), and the Florida Agriculture Statistics Service (FASS), the state of Florida accounts for 65% of the total citrus production in the United States, worth an estimated $ 1.35 billion dollars in revenue per year. In 1997, 254 million boxes of citrus were produced in Florida. This production dropped to 96 million boxes by the year 2014, corresponding to a 62% loss in citrus production in the state of Florida within this period of time. HLB continued to disrupt the citrus industry with an alarming 29% drop in production for the growing season 2

3 Antibiotics to Fight Against Citrus Greening Disease Most commonly used antibiotics Penicillin G Streptomycin Tetracycline Tetracycline, streptomycin, and penicillin have each been shown to be effective in combating this disease. The use of tetracycline began in the 1970s as a promising treatment, but was discontinued as a result of phytotoxicity and high cost. Alternatively, streptomycin was also effective at suppressing the disease, but only when it is applied at a high concentration level. 3

4 Penicillin G to Fight Against Citrus Greening Disease Collaboration with the Division of Plant Industry (DPI). Penicillin G treatment resulted in some reduction of Candidatus Liberibacter asiaticus (Las) populations. Passive injection of penicillin-g demonstrated great effectiveness in reducing Las populations, while penicillin foliar spray has significantly improved tree conditions in a year. 4

5 Treatment of Citrus Trees with Penicillin G Untreated Treated Trees treated with the antibiotic appeared much healthier; with dense new foliage growth and larger, more plentiful fruit

6 Penicillin G Degradation Chatterjee et. al., Current Science. 2004, 87,

7 Penillic, Penilloic, and Penicilloic Acids Mechanisms of Formation Penicillin G acidic hydrolysis Penicillin G basic hydrolysis F. Aldeek et al. Identification of penicillin G metabolites under various environmental conditions using UHPLC- MS/MS. J. Agric. Food. Chem. 2016, ASAP. Manuscript jf v.r2.

8 Possible Metabolites Benzylpenicillin C 16 H 18 N 2 O 4 S, Mw = g/mol Benzylpenicilloic acid C 16 H 20 N 2 O 5 S, Mw = g/mol Benzylpenillic acid C 16 H 18 N 2 O 4 S, Mw = g/mol Benzylpenicillenic acid C 16 H 18 N 2 O 5 S, Mw = g/mol Benzylpenilloic acid C 15 H 20 N 2 O 3 S, Mw = g/mol Penicillamine C 5 H 11 NO 2 S, Mw = g/mol Benzylpenilloaldehyde C 10 H 11 NO 2, Mw = g/mol Aminopenicillanic acid C 8 H 12 N 2 O 3 S, Mw = g/mol Benzylpenamaldic acid C 15 H 18 N 2 O 5 S, Mw = g/mol Benzylpenaldic acid C 10 H 9 NO 4, Mw = g/mol Isopenicillic acid C 16 H 18 N 2 O 4 S, Mw = g/mol F. Aldeek et al. Identification of penicillin G metabolites under various environmental conditions using UHPLC-MS/MS. J. Agric. Food. Chem. 2016, ASAP. Manuscript jf v.r2 8

9 Penicillin G Major Metabolites Penillic acid Penicilloic acid Penilloic acid

10 Penicillin G metabolites Evaluated using Various Analytical Techniques - Hydroxylamine method: To determine penicillin G concentration as a function of ph and temperature, showing that penicillin G is most stable at a ph range of 5-8, and in a temperature range of 0-52 C. - UV-visible spectroscopy: It has proved valuable in evaluating penicillin G degradation by monitoring the development of a 320 nm absorbance peak that arises from the hydrolysis of the β-lactam ring. - UV-visible spectroscopy as a detection method for metabolites in high performance liquid chromatography (HPLC) analysis but this requires high concentrations of analyte, and is unsuitable for the detection of metabolites at the chemical residue level. - HPLC coupled to photodiode array detectors - A few varieties of mass spectrometers have been used for the detection of penicillin G and its metabolites: 1- Single quadrupole for detection of penicillin G and penilloic acid, penicilloic acid, and isopenillic acid in wastewater. 2- linear ion trap to detect new penicillin G metabolites in human serum 3- time-of-flight instruments to detect penicillin in milk 4- triple-quadrapole for - The detection of penicilloic acid in milk - Degradation of penicillin G and two other penicillin antibiotics in acidic condition in the presence of β-lactamase - Determination of the fate of penicillin G and its degradation products in the wastewater from a penicillin G

11 Identification of Penicillin G and its Metabolites UltiMate 3000 UHPLC- Q Exactive Orbitrap (Thermo) F. Aldeek et al. Identification of penicillin G metabolites under various environmental conditions using UHPLC- MS/MS. J. Agric. Food. Chem. 2016, ASAP. Manuscript jf v.r2.

12 Identification of Penicillin G and its Metabolites Water Acquity-6500 Qtrap (SCIEX) F. Aldeek et al. Identification of penicillin G metabolites under various environmental conditions using UHPLC- MS/MS. J. Agric. Food. Chem. 2016, ASAP. Manuscript jf v.r2.

13 Penicillin G Degradation and Metabolites Formation in Various Conditions 100 ng/g ph = 2 ph = 12????? Metabolites????? Metabolites Quantitation of metabolites has been performed using single standards at 100 ng/g

14 Concentration (ng/ml) Penicillin G Degradation and Metabolites Formation in Various Conditions Concentration (ng/ml) Water Acquity-6500 Qtrap (SCIEX) ph 2 A 100 ph 12 Penicillin G Penilloic acid 80 Penillic acid Penicilloic acid 60 B Penicillin G Penillic acid Penilloic acid Penicilloic acid Time (Hours) Time (Hours) - Rapid degradation of penicillin G to completely decomposed within 24hrs - Half life time of penicillin G are 2.4 and 1.35 hours in acidis and basic conditions, respectiverly - Penillic acid is the major product in acidic conditions and not found in alkaline conditions - Penicilloic acid and penilloic acid are found at all ph values, but more in basic conditions 14

15 Concentration (ng/ml)x 100 Penicillin G Degradation and Metabolites Formation in Various Conditions Concentration (ng/ml)x 100 UltiMate 3000 UHPLC- Q Exactive Orbitrap (Thermo) ph 2 A 1.0 ph 12 Penicillin G Penilloic acid Penillic acid Penicilloic acid B Penicillin G Penilloic acid Penillic acid Penicilloic acid Time (Hours) Time (Hours) Results nearly identical between UHPLC-MS/MS platforms

16 Penicillin G Degradation and Metabolites Formation in Lemon Matrix Concentration (ng/ml) x 100 UltiMate 3000 UHPLC- Q Exactive Orbitrap (Thermo) Penicillin G Penilloic acid Penillic acid Penicilloic acid Time (Day) - Lemon matrix measured to be approximately ph 4 - Degradation of penicillin G occurs over 5 days, Half-life time in lemon is 0.9 days - All three metabolites formed, penillic acid is the major product

17 Chemical Kinetics: Degradation Pen G and Formation of Metabolites First-order reaction: [Analyte] = [Analytes] 0 x (1-exp(-kt)) Where: [Analytes] 0 = initial concentration k = rate of degradation or formation of metabolites t = time at which half of penicillin G is degraded Penicillin G degradation ph 2 ph 12 Lemon matrix Rate of degradation 0.29 (hr -1 ) 0.51 (hr -1 ) 0.8 (d -1 ) Half-life 2.4 (hrs) 1.35 (hrs) 0.9 (day) Rate of metabolite formation (hr -1 ) ph 2 ph 12 Lemon matrix Penillic acid 0.38 Not formed 0.9 (d -1 ) Penilloic acid (d -1 ) Penicilloic acid (d -1 )

18 Identification of Metabolites Extracted from a Citrus Field Sample Trees infected with citrus greening disease were treated with penicillin G then harvested after 170 days.

19 Extraction of Penicillin and its Metabolites G From Citrus Commodities (1) (2) (3) (4) (5) (6) (12) (11) (10) (9) (8) (7) 2 g PBS buffer ph = 7 Transfer Remove Hexane Centrifuge Shake for 10 min Add Hexane (13) (14) (15) (16) (17) (18) (19) (20) Shake for 10 min Centrifuge Rinse and centrifuge SPE Trapping in SPE Eluting Dry and reconstitute PVDP filter 19

20 For more details on the analytical and extraction methods used for the identification of penicillin G and its metabolites in citrus samples, please visit our poster

21 F. Aldeek et al. LC-MS/MS method for determination and quantification of penicillin G and its metabolites in citrus fruits affected by Huanglongbing. J. Agric. Food. Chem. 2015, 63,

22 Identification of Metabolites in Citrus Field Sample using the QExactive - Penicillin G was not detected - All three major metabolites were identified - New metabolite, isopenillic acid, was found!

23 Identification of Isopenillic Acid Penillic acid Isopenillic acid min AA: Area = 2.91E6 m/z = Area = 5.95E5 m/z = 335 MA: min Penillic acid Time (min) Isopenillic acid

24 Field Trials Hamlin ( Harvested after 117 Days) Penicillin Penillic Penilloic Tree # (Rate) G (ppb) Acid (ppb) Acid (ppb) 1 (Control) ND ND ND 10 (Control) ND ND ND 18 (Control) ND ND ND 2 (1K) ND (1K) ND (1K) ND (1K) ND (1K) ND (1K) ND (1K) ND (1K) ND (1K) ND (1K) ND (6K) ND (6K) ND (6K) ND (6K) ND (6K) ND (6K) ND (6K) ND (6K) ND (6K) ND (6K) ND (10K) ND BQL = 1 ppb samples samples Hamlin whole fruits ( Harvested after 210 days) Penicillin Penillic Penilloic Tree # (Rate) G (ppb) Acid (ppb) Acid (ppb) 4 (Control) ND ND ND 10 (Control) ND ND ND 11 (Control) ND ND ND 1 (1K) ND BQL (1K) ND ND (1K) ND ND BQL 5 (1K) ND ND (1K) ND ND (1K) ND ND (1K) ND ND BQL 9 (1K) ND BQL (1K) ND ND BQL 13 (1K) ND ND 2.0 Hamlin juice ( Harvested after 210 days) Penicillin Penillic Penilloic Tree # (Rate) G (ppb) Acid (ppb) Acid (ppb) 4 (Control) ND ND ND 10 (Control) ND ND ND 11 (Control) ND ND ND 1 (1K) ND ND ND 2 (1K) ND ND BQL 3 (1K) ND ND ND 5 (1K) ND ND (1K) ND ND ND 7 (1K) ND ND BQL 8 (1K) ND ND ND 9 (1K) ND ND (1K) ND ND ND 13 (1K) ND ND ND

25 F. Aldeek et al. Identification of penicillin G metabolites under various environmental conditions using UHPLC- MS/MS. J. Agric. Food. Chem. 2016, ASAP. Manuscript jf v.r2.

26 Method Updates and New Validation UHPLC-MS/MS Method for the Quantitation of Penicillin G and Metabolites in Citrus Fruit using Internal Standards

27 Standards A B C (1) Penicillin G (2) Penillic acid (3) Penilloic acid Penicilloic acid Aminopenicillanic acid These two standards were not used in the method (1) Penicillin G-D5 (2) Penillic acid-d5 (3) Penilloic acid-d5 27

28 LC-MS/MS Method Validation for the Determination of Penicillin G and its Metabolites using Internal Standards SK9

29 Recoveries with and without Internal Standards Analyte LC-MS recovery (RSD, %) conc. (ng/g) Orange matrix Penicillin G 335>114 Da Penillic acid 335>128 Da Penilloic acid 309>174 Da (4) 56 (11) 58 (12) (4) 65 (4) 61 (4) 1 53 (3) 57 (3) 47 (3) (7) 65 (1) 57 (4) Grapefruit Penicillin G Penillic acid Penilloic acid matrix 335>114 Da 335>128 Da 309>174 Da (1) 74 (3) 74 (5) (13) 68 (8) 68 (10) 1 63 (9) 57 (3) 45 (2) (5) 75 (2) 69 (4) Analyte conc. (ng/g) LC-MS recovery (RSD, %) using IS Orange matrix Penicillin G 335>114 Da Penillic acid 335>128 Da Penilloic acid 309>174 Da (20) 93 (15) 97 (6) (8) 82 (3) 105 (6) (5) 83 (1) 104 (4) (6) 90 (4) 104 (1) Grapefruit Penicillin G Penillic acid Penilloic acid matrix 335>114 Da 335>128 Da 309>174 Da (17) 109 (8) 107 (6) (4) 110 (23) 93 (10) (8) 88 (4) 102 (7) (9) 98 (3) 105 (1)

30 Conclusions - Penicillin G and three of its major metabolites were identified using two different UHPLC-MS/MS platforms, the Q Exactive Orbitrap and the 6500 Qtrap Triple Quardupole. - Penicillin G degrades rapidly under acidic and alkaline conditions, giving rise to penillic acid, penilloic acid, and penicilloic acid. - The chemical kinetics curves of the degradation of penicillin G and formation of metabolites were fitted using first-order reaction. - The three major metabolites have been confirmed using distinct ion products in a citrus sample taken from a tree treated with penicillin G. -A fourth metabolite, isopenillic acid, has also been identified in the field sample. - A new validation method was performed using internal standards of metabolites for better accuracy.

31 Acknowledgments Daniele Canzani Matthew Standland Dr. Mark Crosswhite Dr. Kevin Hsieh Ghislain Gerard Walter Hammack Jo-Marie Cook Amy Brown Mariana Herceg Rebecca Riddle Qi Huanwu - Citrus Plant Greening Foundation for financial support - Dr. Xiaoan Sun, FDACS-DPI Plant Pathologist - Dr. Greg Hodges, FDACS-DPI Chief-Entomology, Nematology and Plant Pathology