Article pubs.acs.org/JAFC
Identification of Penicillin G Metabolites under Various Environmental Conditions Using UHPLC-MS/MS Fadi Aldeek,* Daniele Canzani, Matthew Standland, Mark R. Crosswhite, Walter Hammack, Ghislain Gerard, and Jo-Marie Cook Division of Food Safety, Florida Department of Agriculture and Consumer Services, 3125 Conner Boulevard, Tallahassee, Florida 32399-1650, United States S Supporting Information *
ABSTRACT: In this work, we investigate the stability of penicillin G in various conditions including acidic, alkaline, natural acidic matrices and after treatment of citrus trees that are infected with citrus greening disease. The identification, confirmation, and quantitation of penicillin G and its various metabolites were evaluated using two UHPLC-MS/MS systems with variable capabilities (i.e., Thermo Q Exactive Orbitrap and Sciex 6500 QTrap). Our data show that under acidic and alkaline conditions, penicillin G at 100 ng/mL degrades quickly, with a determined half-life time of approximately 2 h. Penillic acid, penicilloic acid, and penilloic acid are found to be the most abundant metabolites of penicillin G. These major metabolites, along with isopenillic acid, are found when penicillin G is used for treatment of citrus greening infected trees. The findings of this study will provide insight regarding penicillin G residues in agricultural and biological applications. KEYWORDS: penicillin G, stability, degradation, metabolites, identification, liquid chromatography, mass spectrometry
■
INTRODUCTION Due to its high antimicrobial activity, low toxicity, and cost, penicillin G became one of the most widely used antibiotics in a variety of medicinal and agricultural applications.1−5 Penicillin G features a thiazolidine ring, which is fused to a highly labile βlactam ring carbonyl at the 7-position (Figure 1).6,7 Penicillin G is characterized by its susceptibility to ring-opening under various conditions including the presence of acids, bases, nucleophiles, oxidizing agents, heat, UV light, or polar solvents such as water and alcohol.8−10 The highly strained β-lactam ring and its amide bond break in the presence of these conditions to give an array of products (metabolites) such as penicilloic acid, penicillinic acid, penicillamine, penilloaldehyde, aminopenicillanic acid, penaldic acid, penamaldic acid, isopenicillic acid, penillic acid, and penilloic acid (Figure 1).11,12 Penicillin is known to cause allergies for a low percentage of patients. The factors for eliciting allergic reactions have been well studied and found to be caused by the presence of low molecular weight penicillin G metabolites, which are immunogenic.13−15 More importantly, it has been found that these metabolites may bind covalently to biological molecules that are present in any biological system to which penicillin G has been applied. These metabolite−biomolecule conjugates can also be the causative factors for these allergic reactions.16−18 The stability, degradation, and formation of penicillin G metabolites have been evaluated using a variety of analytical techniques. Among them, Khierolomoom et al. have used the hydroxylamine method19 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.20 UV−visible spectroscopy 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.19 Others have © XXXX American Chemical Society
used UV−visible spectroscopy as a detection method for metabolites in high-performance liquid chromatography (HPLC) analysis,21−24 but this requires high concentrations of analyte and is unsuitable for the detection of metabolites at the chemical residue level.24 In addition, several groups have used HPLC coupled to photodiode array detectors.21,25 Although HPLC coupled to mass spectrometry (MS) provides high sensitivity and selectivity compared to the above methods, its use for the determination, identification, and quantitation of penicillin G and its metabolites is still limited. A few varieties of mass spectrometers have been used for the detection of penicillin G and its metabolites.1,26−30 These mass spectrometers include a single quadrupole for the detection of penicillin G and its degradation products (penilloic acid, penicilloic acid, and isopenillic acid) in wastewater,9 a linear ion trap to detect new penicillin G metabolites in human serum,24 time-of-flight instruments to detect penicillin in milk,31 and a triple quadrapole for the detection of penicilloic acid in milk.2 The stability of penicillin G in various conditions and identification of its metabolites using HPLC-MS have been discussed by a few groups. For example, Sun et al. investigated the degradation of penicillin G and two other penicillin antibiotics in acidic condition in the presence of β-lactamase using UHPLC-MS.32 They studied the influence of various parameters including βlactamase dosage, temperature, time, and acidity and found that although penicilloic acid was the dominant species obtained at pH 6 at 40 °C, penilloic acid was obtained at pH 2 above 40 Special Issue: 52nd North American Chemical Residue Workshop Received: December 29, 2015 Revised: February 23, 2016 Accepted: February 24, 2016
A
DOI: 10.1021/acs.jafc.5b06150 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Schematic representation of the chemical structures of pencillin G and its known metabolites. The exact calculated m/z values given are for the protonated molecules.
°C. In another example, Li at al. described the determination of the fate of penicillin G and its degradation products in wastewater from a penicillin G production facility using the same technique.9 They found that the main penicillin G products in surface water were penilloic acid, penicilloic acid, and isopenillic acid. On the basis of the above information, it is worth noting the importance of designing analytical methods that identify not only penicillin G but also these potential metabolites. Recently, we have developed a method for the extraction, identification, and quantitation of penicillin G and two of its metabolites (e.g., penillic acid and penilloic acid) in a variety of citrus fruit using UHPLC-MS/MS. In this method, two product
ion transitions have been used along with ion ratios for data acquisition and analyte confirmation, resulting in a highly selective method with a limit of detection (LOD) of 0.1 ng/g when 2 g of citrus is extracted. In the present paper, we systematically investigated the stability of penicillin G in harsh pH (2−12) conditions, in lemon matrix, and after agricultural application whereby penicillin G was used as a treatment of citrus trees infected by citrus greening disease. This stability was studied using two UHPLC-MS/MS systems with different mass spectrometers, a Thermo Q Exactive Orbitrap and a 6500 QTrap from Sciex. The data obtained using the two systems are quite similar, demonstrating our capability of identifying and quantitating penicillin G and its metabolites using different B
DOI: 10.1021/acs.jafc.5b06150 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
acid, penilloic acid, and penicilloic acid each in PBS buffer at pH 7 were included in the sequence. This was to identify, confirm, and quantitate the metabolite formation after the penicillin degradation by single-point calibration. The stability of the penicillin G in these different buffer solutions was monitored for a period of 24 h. UHPLC-MS/MS Method (1). An Acquity UPLC I-Class System (Waters, Milford, MA, USA) connected to an API 6500 QTrap (Sciex, Toronto, Canada) triple-quadrupole mass spectrometer was used. Using multiple reactions monitoring (MRM), analyses were carried out using electrospray ionization (ESI) in positive-ion mode. Optimized mass spectrometer parameters for the detection of penicillin G and its metabolites were obtained through infusion studies. The parameters optimized were declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP). The MS/MS product ion spectra of the standards and experimental samples were obtained using a target-oriented screening approach in enhanced product ion (EPI) scan mode. The source parameters (ion source voltage (4500 V), curtain gas (30 psi), heater gas (45 psi), and ion source temperature (400 °C)) were identical in MRM and EPI scan modes. Separation of the test compounds was achieved using a 150 mm × 2.1 mm i.d., 1.7 μm, Acquity C18 column. The mobile phase composition was (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The column temperature was maintained at 40 (±1) °C. The gradient began at 95:5 of A:B with a flow of 0.2 mL/min. The gradient was changed to 5:95 of A:B over a course of 10 min followed by a 5 min hold at 5:95 of A:B. Over 0.1 min, the gradient was changed to 95:5 of A:B with a concomitant change in flow rate to 0.4 mL/min. After 4 min, the flow was reduced to 0.2 mL/min over the course of 0.1 min. This was held for 0.8 min to complete the LC gradient (20 min total run time). Instrument control and data acquisition and evaluation were performed with the AB Sciex Analyst 1.6.2 software. UHPLC-MS/MS Method (2). For separation, a Thermo/Dionex UltiMate 3000 UHPLC was fitted with a Waters Acquity 150 mm C18 column with a 2.1 mm i.d. and 1.7 μm packing. The mobile phase composition was (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The column temperature was maintained at 40 °C. This LC system was coupled to a Thermo Q Exactive mass spectrometer for analysis, using an electrospray ionization source in positive-ion mode, maintained at 300 °C. A 10 min elution gradient was chosen because it was sufficient to separate penilloic acid and penicilloic acid and keep their respective isomers unresolved. The gradient began at 95:5 of A:B with a flow of 0.2 mL/min. The gradient was changed to 5:95 of A:B over a course of 10 min followed by a 5 min hold at 5:95 of A:B. Over 0.1 min, the gradient was changed to 95:5 of A:B. The total run time was 20 min. Full scan mode from m/z 100 to 1000 was used to detect the 335.1060 amu parent masses of penicillin G and penillic acid, and the 15 and 30 CE filters were used for the detection of their product ions. Because the structures of penicilloic acid and penilloic acid (Figure 1) differ in only one carboxyl group, they elute within 0.1−0.2 min of each other and produce overlapping and nearly identical mass spectra. To clearly distinguish between the two compounds, the parallel reaction monitoring (PRM) mode of the Q Exactive was used to filter both the 353.1165 and 309.1267 amu parent masses. After isolation of the parents, the 30 CE filter was applied, producing unique ion products for both compounds. Instrument control and data acquisition and evaluation were performed with Thermo Xcaliber software.
UHPLC-MS/MS instruments. In addition, the data show that penicillin G degrades very quickly, depending on the studied conditions, leading to the formation of a series of abundant metabolites (e.g., penillic acid, penilloic acid, penicilloic acid, and isopenillic acid).
■
MATERIALS AND METHODS
Reagents. Penilloic acid (96.3%), penillic acid (99.3%), and penicilloic acid (91.9%) were purchased from LGC Standards (Wesel, Germany). Penicillin G potassium salt (99.4%) was provided by o2si Smart Solutions (Charleston, SC, USA). UHPLC grade hexane, acetonitrile (with and without 0.1% formic acid), methanol, and water (with and without 0.1% formic acid) were provided by Fisher Scientific. Anhydrous monosodium phosphate, disodium phosphate, and ammonium acetate were purchased from Sigma-Aldrich. The phosphate buffer solution (PBS) (0.1 M, pH 7) was prepared by dissolving anhydrous NaH2PO4 (5.8 g, 0.0483 mol) and Na2HPO4 (8.15 g, 0.0574 mol) in 1 L of DI water. Preparation of Standard Stock Solutions. Individual stock solutions of penicillin G potassium salt, penilloic acid, and penillic acid at 1000 μg/mL were made by dissolving the standard (after adjustment for salt content and purity) in a mixture of water and acetonitrile (1:1, v/v), whereas individual stock solution of penicilloic acid at 1000 μg/mL was made by first dissolving the standard with 300 μL of DMSO followed by the addition of phosphate buffer. These stock standard solutions were stored at −20 °C for an extended period of time (>5 months) with no degradation. These stock solutions, each at 1000 μg/mL, were utilized to prepare 1 μg/mL standard solutions of each compound. These were prepared by adding 100 μL of each individual stock solution into the appropriate 100 mL volumetric flask and filling each flask to the mark with phosphate buffer (0.1 M, pH 7) to produce a solution with a concentration of 1 μg/mL for each compound. Preparation of Lemon Matrix. Lemon sample was stored at −80 °C. The frozen whole fruits were broken into smaller pieces and then homogenized into a powder using a Blixer RSI BX6 industrial food processor (Robot Coupe, Ridgeland, MS, USA). Two grams (±0.05 g) of the homogenized fruit was immediately weighed into a 50 mL polypropylene centrifuge tube (Fisherbrand, Pittsburgh, PA, USA), followed by the addition of 4 mL of PBS buffer (0.1 M, pH 7). The sample was then shaken at 1100 rpm for 10 min in a SPEX Sample Prep Geno/Grinder. Then, 4 mL of hexane was added, and the sample was shaken again at 1100 rpm for 10 min. The sample was then centrifuged at 4000 rpm for 15 min to separate the aqueous and organic layers. The hexane layer was removed using a Pasteur pipet, and the aqueous layer was transferred into a Milipore Amicron Ultra 50000 Mw membrane filtration device (Millipore, Darmstadt, Germany). Residual hexane and remaining solids were removed by centrifugation at 4000 rpm for 15 min. The pH of the citrus filtrate was then measured using pH strips and was found to be around 4. UHPLC-MS/MS Analysis of Penicillin G Degradation in Lemon Matrix. Nine hundred microliters of lemon matrix was added into LC vials and spiked with 100 μL of 1000 ppb penicillin G, penillic acid, penilloic acid, or penicilloic acid stock solutions. Here, the penilloic acid, penillic acid, and penicilloic acid in matrix were used for identification, confirmation, and quantitation purposes. These solutions have been injected on the UHPLC-MS/MS instruments (API 6500 QTrap, Sciex, Toronto, ON, Canada, and Q Exactive Orbitrap, Thermo, Waltham, MA, USA) every day for a week. Preparation of PBS at Different pH Values. Ten milliliters of PBS buffer solutions at pH 2 and 12 were made by adjusting the above PBS buffer stock (0.1 M pH 7) using 0.1 M NaOH or 0.1 M HCl solutions. Penicillin G Stability at Various pH Values. Nine hundred microliters of each buffer solution at pH 2 and 12 was added to individual LC vials, followed by the addition of 100 μL of penicillin G (1000 ng/mL) solution to each vial approximately 10 min before the UHPLC-MS/MS injection to prevent penicillin G degradation prior to analysis. Additionally, 100 ng/mL solutions of penicillin G, penillic
■
RESULTS AND DISCUSSION The unstable β-lactam ring contained within penicillin G is highly susceptible to hydrolytic opening under a variety of conditions including heat, acidic or alkaline conditions, and under enzymatic activity.9,20 Upon penicillin G exposure to these conditions, the opening of the β-lactam function will lead to a variety of byproducts, known as metabolites. Figure 1 represents most of the metabolites that have been illustrated in the literature. For example, in highly acidic or alkaline media, C
DOI: 10.1021/acs.jafc.5b06150 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry hydrolysis reaction occurs, leading to the formation of penillic acid or penilloic acid and penicilloic acid, respectively.8,33 Figure 2 shows a schematic representation of the hydrolysis mechanism involved for the formation of the aforementioned metabolites. It is worth mentioning that on the basis of our
extensive literature research, these three metabolites seem to be the most abundant. Penillic acid can be converted into isopenillic acid in alkaline or anaerobic conditions. 34 Furthermore, a hydrolysis reaction may occur on the amide function present in the penicillin G molecular structure, yielding metabolites such as penicillamine and penilloaldehyde.9,12 Alternatively, the removal of the acyl side chain through enzymatic activity leads to the formation of 6aminopenicillanic acid.10 Analysis of Penicillin G and Metabolites Using UHPLC-MS/MS. Solutions (0.1 ng/μL) of penicillin G, penillic acid, penilloic acid, and penicilloic acid and a mix of these four compounds were prepared and independently injected on the Acquity UPLC I-Class System connected to an API 6500 QTrap and on an Ultimate 3000 UHPLC connected to a Q Exactive orbitrap. Extracted chromatograms for each of the four compounds analyzed are shown in Figures 3 and 4 for the 6500 QTrap and the Q Exactive, respectively. Typical retention times of 6.61, 5.20, 5.10, and 4.24 min and 7.08, 5.84, 5.63, and 4.82 min are reported for penicillin G, penicilloic acid, penilloic acid, and penillic acid when the 6500 QTrap instrument and Q Exactive are used, respectively. These retention times were expected on the basis of polarity of these compounds. The shortest retention for penillic acid is explained by the high polarity of this compound bearing two carboxylic acid groups and imidazoline heterocycle in its structure. On the other hand, penicillin G features only one carboxylic acid group along with a nonpolar benzyl group contributing to its longer retention time of 6.61 min (Figure 1). However, the retention time of penicilloic acid was found to be 0.1−0.2 min shorter than that of penilloic acid due to the presence of an additional carboxylic acid function. The product ion transitions used for quantification and confirmation were selected on the basis of both the sensitivity and the presence of a clean background (Figures 3 and 4). Three transitions have been selected for each analyte for their consistency of ion ratios RSD of
Identification of Penicillin G Metabolites under Various Environmental Conditions Using UHPLC-MS/MS Fadi Aldeek,* Daniele Canzani, Matthew Standland, Mark R. Crosswhite, Walter Hammack, Ghislain Gerard, and Jo-Marie Cook Division of Food Safety, Florida Department of Agriculture and Consumer Services, 3125 Conner Boulevard, Tallahassee, Florida 32399-1650, United States S Supporting Information *
ABSTRACT: In this work, we investigate the stability of penicillin G in various conditions including acidic, alkaline, natural acidic matrices and after treatment of citrus trees that are infected with citrus greening disease. The identification, confirmation, and quantitation of penicillin G and its various metabolites were evaluated using two UHPLC-MS/MS systems with variable capabilities (i.e., Thermo Q Exactive Orbitrap and Sciex 6500 QTrap). Our data show that under acidic and alkaline conditions, penicillin G at 100 ng/mL degrades quickly, with a determined half-life time of approximately 2 h. Penillic acid, penicilloic acid, and penilloic acid are found to be the most abundant metabolites of penicillin G. These major metabolites, along with isopenillic acid, are found when penicillin G is used for treatment of citrus greening infected trees. The findings of this study will provide insight regarding penicillin G residues in agricultural and biological applications. KEYWORDS: penicillin G, stability, degradation, metabolites, identification, liquid chromatography, mass spectrometry
■
INTRODUCTION Due to its high antimicrobial activity, low toxicity, and cost, penicillin G became one of the most widely used antibiotics in a variety of medicinal and agricultural applications.1−5 Penicillin G features a thiazolidine ring, which is fused to a highly labile βlactam ring carbonyl at the 7-position (Figure 1).6,7 Penicillin G is characterized by its susceptibility to ring-opening under various conditions including the presence of acids, bases, nucleophiles, oxidizing agents, heat, UV light, or polar solvents such as water and alcohol.8−10 The highly strained β-lactam ring and its amide bond break in the presence of these conditions to give an array of products (metabolites) such as penicilloic acid, penicillinic acid, penicillamine, penilloaldehyde, aminopenicillanic acid, penaldic acid, penamaldic acid, isopenicillic acid, penillic acid, and penilloic acid (Figure 1).11,12 Penicillin is known to cause allergies for a low percentage of patients. The factors for eliciting allergic reactions have been well studied and found to be caused by the presence of low molecular weight penicillin G metabolites, which are immunogenic.13−15 More importantly, it has been found that these metabolites may bind covalently to biological molecules that are present in any biological system to which penicillin G has been applied. These metabolite−biomolecule conjugates can also be the causative factors for these allergic reactions.16−18 The stability, degradation, and formation of penicillin G metabolites have been evaluated using a variety of analytical techniques. Among them, Khierolomoom et al. have used the hydroxylamine method19 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.20 UV−visible spectroscopy 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.19 Others have © XXXX American Chemical Society
used UV−visible spectroscopy as a detection method for metabolites in high-performance liquid chromatography (HPLC) analysis,21−24 but this requires high concentrations of analyte and is unsuitable for the detection of metabolites at the chemical residue level.24 In addition, several groups have used HPLC coupled to photodiode array detectors.21,25 Although HPLC coupled to mass spectrometry (MS) provides high sensitivity and selectivity compared to the above methods, its use for the determination, identification, and quantitation of penicillin G and its metabolites is still limited. A few varieties of mass spectrometers have been used for the detection of penicillin G and its metabolites.1,26−30 These mass spectrometers include a single quadrupole for the detection of penicillin G and its degradation products (penilloic acid, penicilloic acid, and isopenillic acid) in wastewater,9 a linear ion trap to detect new penicillin G metabolites in human serum,24 time-of-flight instruments to detect penicillin in milk,31 and a triple quadrapole for the detection of penicilloic acid in milk.2 The stability of penicillin G in various conditions and identification of its metabolites using HPLC-MS have been discussed by a few groups. For example, Sun et al. investigated the degradation of penicillin G and two other penicillin antibiotics in acidic condition in the presence of β-lactamase using UHPLC-MS.32 They studied the influence of various parameters including βlactamase dosage, temperature, time, and acidity and found that although penicilloic acid was the dominant species obtained at pH 6 at 40 °C, penilloic acid was obtained at pH 2 above 40 Special Issue: 52nd North American Chemical Residue Workshop Received: December 29, 2015 Revised: February 23, 2016 Accepted: February 24, 2016
A
DOI: 10.1021/acs.jafc.5b06150 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Schematic representation of the chemical structures of pencillin G and its known metabolites. The exact calculated m/z values given are for the protonated molecules.
°C. In another example, Li at al. described the determination of the fate of penicillin G and its degradation products in wastewater from a penicillin G production facility using the same technique.9 They found that the main penicillin G products in surface water were penilloic acid, penicilloic acid, and isopenillic acid. On the basis of the above information, it is worth noting the importance of designing analytical methods that identify not only penicillin G but also these potential metabolites. Recently, we have developed a method for the extraction, identification, and quantitation of penicillin G and two of its metabolites (e.g., penillic acid and penilloic acid) in a variety of citrus fruit using UHPLC-MS/MS. In this method, two product
ion transitions have been used along with ion ratios for data acquisition and analyte confirmation, resulting in a highly selective method with a limit of detection (LOD) of 0.1 ng/g when 2 g of citrus is extracted. In the present paper, we systematically investigated the stability of penicillin G in harsh pH (2−12) conditions, in lemon matrix, and after agricultural application whereby penicillin G was used as a treatment of citrus trees infected by citrus greening disease. This stability was studied using two UHPLC-MS/MS systems with different mass spectrometers, a Thermo Q Exactive Orbitrap and a 6500 QTrap from Sciex. The data obtained using the two systems are quite similar, demonstrating our capability of identifying and quantitating penicillin G and its metabolites using different B
DOI: 10.1021/acs.jafc.5b06150 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
acid, penilloic acid, and penicilloic acid each in PBS buffer at pH 7 were included in the sequence. This was to identify, confirm, and quantitate the metabolite formation after the penicillin degradation by single-point calibration. The stability of the penicillin G in these different buffer solutions was monitored for a period of 24 h. UHPLC-MS/MS Method (1). An Acquity UPLC I-Class System (Waters, Milford, MA, USA) connected to an API 6500 QTrap (Sciex, Toronto, Canada) triple-quadrupole mass spectrometer was used. Using multiple reactions monitoring (MRM), analyses were carried out using electrospray ionization (ESI) in positive-ion mode. Optimized mass spectrometer parameters for the detection of penicillin G and its metabolites were obtained through infusion studies. The parameters optimized were declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP). The MS/MS product ion spectra of the standards and experimental samples were obtained using a target-oriented screening approach in enhanced product ion (EPI) scan mode. The source parameters (ion source voltage (4500 V), curtain gas (30 psi), heater gas (45 psi), and ion source temperature (400 °C)) were identical in MRM and EPI scan modes. Separation of the test compounds was achieved using a 150 mm × 2.1 mm i.d., 1.7 μm, Acquity C18 column. The mobile phase composition was (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The column temperature was maintained at 40 (±1) °C. The gradient began at 95:5 of A:B with a flow of 0.2 mL/min. The gradient was changed to 5:95 of A:B over a course of 10 min followed by a 5 min hold at 5:95 of A:B. Over 0.1 min, the gradient was changed to 95:5 of A:B with a concomitant change in flow rate to 0.4 mL/min. After 4 min, the flow was reduced to 0.2 mL/min over the course of 0.1 min. This was held for 0.8 min to complete the LC gradient (20 min total run time). Instrument control and data acquisition and evaluation were performed with the AB Sciex Analyst 1.6.2 software. UHPLC-MS/MS Method (2). For separation, a Thermo/Dionex UltiMate 3000 UHPLC was fitted with a Waters Acquity 150 mm C18 column with a 2.1 mm i.d. and 1.7 μm packing. The mobile phase composition was (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The column temperature was maintained at 40 °C. This LC system was coupled to a Thermo Q Exactive mass spectrometer for analysis, using an electrospray ionization source in positive-ion mode, maintained at 300 °C. A 10 min elution gradient was chosen because it was sufficient to separate penilloic acid and penicilloic acid and keep their respective isomers unresolved. The gradient began at 95:5 of A:B with a flow of 0.2 mL/min. The gradient was changed to 5:95 of A:B over a course of 10 min followed by a 5 min hold at 5:95 of A:B. Over 0.1 min, the gradient was changed to 95:5 of A:B. The total run time was 20 min. Full scan mode from m/z 100 to 1000 was used to detect the 335.1060 amu parent masses of penicillin G and penillic acid, and the 15 and 30 CE filters were used for the detection of their product ions. Because the structures of penicilloic acid and penilloic acid (Figure 1) differ in only one carboxyl group, they elute within 0.1−0.2 min of each other and produce overlapping and nearly identical mass spectra. To clearly distinguish between the two compounds, the parallel reaction monitoring (PRM) mode of the Q Exactive was used to filter both the 353.1165 and 309.1267 amu parent masses. After isolation of the parents, the 30 CE filter was applied, producing unique ion products for both compounds. Instrument control and data acquisition and evaluation were performed with Thermo Xcaliber software.
UHPLC-MS/MS instruments. In addition, the data show that penicillin G degrades very quickly, depending on the studied conditions, leading to the formation of a series of abundant metabolites (e.g., penillic acid, penilloic acid, penicilloic acid, and isopenillic acid).
■
MATERIALS AND METHODS
Reagents. Penilloic acid (96.3%), penillic acid (99.3%), and penicilloic acid (91.9%) were purchased from LGC Standards (Wesel, Germany). Penicillin G potassium salt (99.4%) was provided by o2si Smart Solutions (Charleston, SC, USA). UHPLC grade hexane, acetonitrile (with and without 0.1% formic acid), methanol, and water (with and without 0.1% formic acid) were provided by Fisher Scientific. Anhydrous monosodium phosphate, disodium phosphate, and ammonium acetate were purchased from Sigma-Aldrich. The phosphate buffer solution (PBS) (0.1 M, pH 7) was prepared by dissolving anhydrous NaH2PO4 (5.8 g, 0.0483 mol) and Na2HPO4 (8.15 g, 0.0574 mol) in 1 L of DI water. Preparation of Standard Stock Solutions. Individual stock solutions of penicillin G potassium salt, penilloic acid, and penillic acid at 1000 μg/mL were made by dissolving the standard (after adjustment for salt content and purity) in a mixture of water and acetonitrile (1:1, v/v), whereas individual stock solution of penicilloic acid at 1000 μg/mL was made by first dissolving the standard with 300 μL of DMSO followed by the addition of phosphate buffer. These stock standard solutions were stored at −20 °C for an extended period of time (>5 months) with no degradation. These stock solutions, each at 1000 μg/mL, were utilized to prepare 1 μg/mL standard solutions of each compound. These were prepared by adding 100 μL of each individual stock solution into the appropriate 100 mL volumetric flask and filling each flask to the mark with phosphate buffer (0.1 M, pH 7) to produce a solution with a concentration of 1 μg/mL for each compound. Preparation of Lemon Matrix. Lemon sample was stored at −80 °C. The frozen whole fruits were broken into smaller pieces and then homogenized into a powder using a Blixer RSI BX6 industrial food processor (Robot Coupe, Ridgeland, MS, USA). Two grams (±0.05 g) of the homogenized fruit was immediately weighed into a 50 mL polypropylene centrifuge tube (Fisherbrand, Pittsburgh, PA, USA), followed by the addition of 4 mL of PBS buffer (0.1 M, pH 7). The sample was then shaken at 1100 rpm for 10 min in a SPEX Sample Prep Geno/Grinder. Then, 4 mL of hexane was added, and the sample was shaken again at 1100 rpm for 10 min. The sample was then centrifuged at 4000 rpm for 15 min to separate the aqueous and organic layers. The hexane layer was removed using a Pasteur pipet, and the aqueous layer was transferred into a Milipore Amicron Ultra 50000 Mw membrane filtration device (Millipore, Darmstadt, Germany). Residual hexane and remaining solids were removed by centrifugation at 4000 rpm for 15 min. The pH of the citrus filtrate was then measured using pH strips and was found to be around 4. UHPLC-MS/MS Analysis of Penicillin G Degradation in Lemon Matrix. Nine hundred microliters of lemon matrix was added into LC vials and spiked with 100 μL of 1000 ppb penicillin G, penillic acid, penilloic acid, or penicilloic acid stock solutions. Here, the penilloic acid, penillic acid, and penicilloic acid in matrix were used for identification, confirmation, and quantitation purposes. These solutions have been injected on the UHPLC-MS/MS instruments (API 6500 QTrap, Sciex, Toronto, ON, Canada, and Q Exactive Orbitrap, Thermo, Waltham, MA, USA) every day for a week. Preparation of PBS at Different pH Values. Ten milliliters of PBS buffer solutions at pH 2 and 12 were made by adjusting the above PBS buffer stock (0.1 M pH 7) using 0.1 M NaOH or 0.1 M HCl solutions. Penicillin G Stability at Various pH Values. Nine hundred microliters of each buffer solution at pH 2 and 12 was added to individual LC vials, followed by the addition of 100 μL of penicillin G (1000 ng/mL) solution to each vial approximately 10 min before the UHPLC-MS/MS injection to prevent penicillin G degradation prior to analysis. Additionally, 100 ng/mL solutions of penicillin G, penillic
■
RESULTS AND DISCUSSION The unstable β-lactam ring contained within penicillin G is highly susceptible to hydrolytic opening under a variety of conditions including heat, acidic or alkaline conditions, and under enzymatic activity.9,20 Upon penicillin G exposure to these conditions, the opening of the β-lactam function will lead to a variety of byproducts, known as metabolites. Figure 1 represents most of the metabolites that have been illustrated in the literature. For example, in highly acidic or alkaline media, C
DOI: 10.1021/acs.jafc.5b06150 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry hydrolysis reaction occurs, leading to the formation of penillic acid or penilloic acid and penicilloic acid, respectively.8,33 Figure 2 shows a schematic representation of the hydrolysis mechanism involved for the formation of the aforementioned metabolites. It is worth mentioning that on the basis of our
extensive literature research, these three metabolites seem to be the most abundant. Penillic acid can be converted into isopenillic acid in alkaline or anaerobic conditions. 34 Furthermore, a hydrolysis reaction may occur on the amide function present in the penicillin G molecular structure, yielding metabolites such as penicillamine and penilloaldehyde.9,12 Alternatively, the removal of the acyl side chain through enzymatic activity leads to the formation of 6aminopenicillanic acid.10 Analysis of Penicillin G and Metabolites Using UHPLC-MS/MS. Solutions (0.1 ng/μL) of penicillin G, penillic acid, penilloic acid, and penicilloic acid and a mix of these four compounds were prepared and independently injected on the Acquity UPLC I-Class System connected to an API 6500 QTrap and on an Ultimate 3000 UHPLC connected to a Q Exactive orbitrap. Extracted chromatograms for each of the four compounds analyzed are shown in Figures 3 and 4 for the 6500 QTrap and the Q Exactive, respectively. Typical retention times of 6.61, 5.20, 5.10, and 4.24 min and 7.08, 5.84, 5.63, and 4.82 min are reported for penicillin G, penicilloic acid, penilloic acid, and penillic acid when the 6500 QTrap instrument and Q Exactive are used, respectively. These retention times were expected on the basis of polarity of these compounds. The shortest retention for penillic acid is explained by the high polarity of this compound bearing two carboxylic acid groups and imidazoline heterocycle in its structure. On the other hand, penicillin G features only one carboxylic acid group along with a nonpolar benzyl group contributing to its longer retention time of 6.61 min (Figure 1). However, the retention time of penicilloic acid was found to be 0.1−0.2 min shorter than that of penilloic acid due to the presence of an additional carboxylic acid function. The product ion transitions used for quantification and confirmation were selected on the basis of both the sensitivity and the presence of a clean background (Figures 3 and 4). Three transitions have been selected for each analyte for their consistency of ion ratios RSD of
