Simultaneous Estimation of Ampicillin and Sulbactam in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry

Srinivasa Reddy, Licto Thomas, Venkatesan P, Arindam Mukhopadhyay*, Saral Thangam

Norwich Clinical Services, Bangalore, Karnataka, India

*Corresponding Author E-mail: Arindam.Mukhopadhyay@norwichclinical.com

ABSTRACT:

A LCMS/MS method for the simultaneous determination of ampicillin and sulbactam in human plasma was described. After protein precipitation using 2mL of acetonitrile, 250µL of supernatant was mixed with 1.000 mL of 0.1% Acetic Acid in Milli-Q-water. 10µL was injected to a Biobasic AX column and eluted with 10mM Ammonium acetate and Acetonitrile: 60:40, v/v at a flow rate of 0.5mL/min. MRM transitions were monitored in negative mode as m/z 348.1 → 206.8 (AMP), 231.9 → 187.8 (SUL) and m/z 353.0 → 211.9 (AMP D5). Sample concentrations were calculated by linear regression analysis using the analyst software1.6.3. An excellent linear response was obtained over the concentration ranges 0.1040µg/mL to 10.1562µg/mL for Ampicillin and 0.0510µg/mL to 6.1552µg/mL for Sulbactam. The intra-day and inter-day precision were within 3.50% for all analytes. The assay accuracy was 96.27 –103.59 %. Mean recoveries were 84.51% and 98.54% for ampicillin and sulbactam, respectively. The limits of detections were 0.026µg/mL and 0.013µg/mL for ampicillin and sulbactam. This method was successfully used for a bioequivalence study.

KEYWORDS: Ampicillin, sulbactam, antibiotics, LCMS/MS, validation.

INTRODUCTION:

Ampicillin is a broad-spectrum β-lactam antibiotic which is widely used for the treatment of several infections. However, many organisms which were initially sensitive to these penicillins became resistance due to their ability to produce β-lactamases. These enzymes hydrolyse the β-lactam ring and, therefore, destroy the antimicrobial activity of the antibiotics. Sulbactam being a β-lactam agent without significant intrinsic antimicrobial activity acts as an irreversible non-competitive inhibitor of bacterial β -lactamase.

Combinations of β -lactams with β -lactamase inhibitors extend the spectrum of β -lactams by preventing the hydrolysis by β-lactamases. Actually the β -lactam/ lactamase inhibitor combinations are less potent β-lactamase inducers than cephalosporins make them a useful weapon in controlling resistance. Ampicillin/Sulbactam combination (AMP: SUL = 2:1 w/w), which was developed and marketed in the US in 1987, is the most frequently used although both agents do not synergism. It is indicated for the treatment of intra-abdominal, skin and skin structure, and gynaecological infections in adults and is effective against various microorganisms, including β-lactamase-producing Escherichia coli, Klebsiella species, Staphylococcus aureus, and Bacteroides species.^1,2

Several chromatographic methods are available for determination of ampicillin, individually and along with other related compounds.^3-7. However, the difference in the chemical structures of these two compounds and the presence of the doubled amount of ampicillin in the formulation lead to difficulties of simultaneous determination of ampicillin and sulbactam. To investigate the pharmacokinetics of ampicillin/sulbactam (2:1) combination after intravenous administration, concentrations of ampicillin and sulbactam were determined using HPLC or applying post-column reaction technique which is complicated for routine use.^8,9. Even HPLC methods used for routine drug quality control test of ampicillin/sulbactam (2:1) powder for injections have some disadvantages when being applied in such as (1) high back pressure, which reduce the lifetime of analytical column, (2) abnormal tailing and broadening peak shapes, (3) requiring large volumes of organic solvents, (4) long system stabilization time¹⁰. Nahata et al¹ reported a method for simultaneous estimation of ampicillin and sulbactam which utilized protein precipitation method; however, this method requires a post-column derivatization with 2 N sodium hydroxide and an aqueous methanolic solution of mercuric chloride. The limit of quantitation of the method was 0.5mg/mL for sulbactam and 1.0mg/mL for ampicillin. Nalbant et al¹¹ had recently described the simultaneous estimation of ampicillin and sulbactam; however, for both ampicillin and sulbactam, the lower limit of quantitation (LLOQ) was established as 0.25 μg/mL. Since the amount of sulbactam in the drug product is only half of the ampicillin, our objective is to develop a more sensitive method for simultaneously quantitation of ampicillin and sulbactam in plasma which can be used for PK analysis in bio-equivalence studies.

We developed a new, sensitive and relatively simple LC–MS/MS method for simultaneous estimation of ampicillin/sulbactam in human plasma. This method is validated as per FDA regulations and can be used for pharmacokinetic study.¹²

MATERIALS AND METHODS:

Ampicillin Trihydrate (purity: 98.97%) and Sulbactam Sodium (purity: 98.84%) were purchased from BioOrganics, India. Ampicillin-d5 (purity: 97.36%) used as an internal standard was also from BioOrganics, India.

Methanol (HPLC-grade), acetonitrile, ammonium acetate and formic acid of highest purity grade were purchased locally. In this study Milli Q purified water (Millipore, Milford, MA) was used.

Plasma lots collected in house were used for the experiments.

Preparation of analytes and internal standard solutions:

Stock solution of Ampicillin trihydrate (2000µg/mL) was prepared in Milli-Q-water containing 10% DMSO and 1% Acetic Acid. The stock solution of Ampicillin was then diluted with Methanol: Milli-Q-Water: 50:50 (v/v) containing 1% acetic acid to concentration ranges of 5.0056µg/mL to 501.6049µg/mL.

Similarly, Sulbactam sodium stock solution (2000 µg/mL) in Milli-Q-water containing 1% Acetic acid was prepared and then diluted to the concentration range 2.48 µg/mL to 304.87 µg/mL using Methanol: Milli-Q-Water: 50:50 (v/v) containing 1% acetic acid as diluent.

The internal standard, Ampicillin-d5, stock (1000µg/mL) prepared by dissolving in Milli-Q-water containing 1% Acetic acid was then diluted to 2.00µg/mL using diluent as mentioned above.

The concentration of each stock solution was corrected by considering its potency and actual amount weighed before dilution.

Preparation of calibration standards:

To prepare calibration curve standards, 20µL of the diluted samples of each analyte was added to 960µL of K₂EDTA plasma to obtain a concentration range about 0.1001µg/mL to 10.0321µg/mL for Ampicillin and 0.0496µg/mL to 6.0974µg/mL for Sulbactam. All these bulk spiked samples were stored at about -70°C in aliquot of 200µL.

Preparation of Quality Control Samples:

Stock solution of Ampicillin was diluted with Methanol: Milli-Q-Water: 50:50 (v/v) containing 1% acetic acid to obtain the concentration ranges of 5.0115µg/mL to 387.9817µg/mL. Similarly, Sulbactam stock solution was diluted to concentration range of 2.5135µg/mL to 234.6839µg/mL using the same diluent. 20 µL of each diluted solution was then added into 960µL of K₂EDTA plasma to obtain final concentration range about 0.1002µg/mL to 7.7596µg/mL for Ampicillin and 0.0503µg/mL to 4.6937µg/mL for Sulbactam. The samples were then stored at about -70°C.

Sample preparation:

50µL of internal standard mixture (Ampicillin D5) was added to all RIA vials except blank. 100µL of sample was then added to each labeled RIA vial. 2mL of Acetonitrile was then added to the RIA vials, capped them, and then placed on Vibramax at 2500 RPM for 10 mins. They were centrifuged at 11000rpm for 5 mins at about 4°C. 250µL of supernatant from each vial was transferred into fresh RIA vial. 1.000mL of 0.1% Acetic Acid in Milli-Q-water was added to all samples and vortexed. The samples from each vial were then transferred into a labeled HPLC vial and placed in the autosampler.

Table 1: MS parameters optimized for analytes and internal standards

Analyte/IS	Declustering Potential (DP) (V)	Entrance Potential (EP) (V)	Collision Energy (CE) (V)	Collision Cell Exit Potential (CXP) (V)	Collision activated dissociation (CAD) (psi)	Dwell Time (ms)	Ion source voltage (V)	Curtain gas flow (CUR) (psi)
Ampicillin	33	10	16	5	8	200	4500	30
Sulbactam	42	10	15	18	8	200	4500	30
Ampicillin D5	33	10	16	5	8	200	4500	30

Chromatography:

10µL of sample was injected on a Biobasic AX column (100 x 2.1mm, 5µm). Freshly prepared 10mM Ammonium acetate and Acetonitrile: 60:40, v/v was used as a mobile phase at a flow rate of 0.500mL/min without splitter in Waters UPLC attached to API 4000 Mass spectrometer (Applied Biosystems, USA). The column was maintained at 50°C in the column oven. The run time was 3.0 minutes.

Mass Spectrometry:

Electrospray ionization (ESI) interface operated in negative atmospheric pressure ionization mode (API) was used for the multiple reaction monitoring (MRM). The operational conditions were optimized by infusing diluted stock solution of analyte and internal standard (Table 1).

Source temperature was set at 500°C. Nebulizer gas (GS1) and auxiliary gas (GS2) flows were 45 and 55 psi, respectively. Quadrupoles Q1 and Q3 were set on unit resolution.

MRM transitions were monitored as m/z 348.1 → 206.8 (AMP), 231.9 → 187.8 (SUL) and m/z 353.0 → 211.9 (AMP D5).

Sample concentrations were calculated by linear regression analysis using the analyst software 1.6.3. Data was processed by peak area ratio. The concentration of unknown was calculated from the equation (Y= mX+ c) using regression analysis of spiked plasma calibration standards with reciprocal of the square of the drug concentration (1/X²).

RESULTS AND DISCUSSION:

Method Development:

Specific and effective sample clean-up procedures are required for sensitive and selective LC–MS/MS assays for determination of very low concentration levels of pharmaceutical targets present in biological samples. Three methods e.g. protein precipitation (PPT), liquid–liquid extraction (LLE) and solid-phase extraction (SPE) are generally used for preparing biological specimen. SPE technique for sample extraction is good but with an added cost. LLE is comparatively a cumbersome method. Protein precipitation method using organic solvent is the simplest one and is the method of choice for our sample extraction. This technique was shown to be robust, provided clean samples and gave good and reproducible recoveries of both analytes and IS. The extraction recoveries of analytes were determined by comparing peak areas from plasma samples (n = 6) spiked before extraction with those from aqueous samples. The mean recoveries across QC levels (with precision) were 84.51% for ampicillin and 98.54% for sulbactam.

To make the method simpler, we used isocratic mobile phase for eluting the analyte and IS. The total run time was only 3 minutes. A short run time is ideally required for being considered in high throughput analysis. The retention times for ampicillin, sulbactam and IS were 1.41 min, 1.58 min and 1.41 min, respectively.

Method Validation:

FDA Guidelines for specificity, linearity, intra- and inter-day precision and accuracy, and stability were followed to validate this method.¹²

Selectivity:

Selectivity of the method was evaluated in eight individual human plasma lots along with one lipemic and one hemolytic lot. No interference was observed at the retention times of analyte and internal standard when peak responses in blank lots were compared against the response of spiked LLOQ containing IS mixtures. Representative chromatograms in Figures 1(a and b; blank plasma) and 2 (a and b; blank plasma spiked with ampicillin-Fig 2a and Sulbactam-Fig 2b at LLOQs and IS) demonstrated the selectivity of the method. The minimum signal to noise ratios were 2142.49 for Ampicillin and 327.80 for Sulbactam (more than 5 is acceptable).

Linearity and Sensitivity:

Eight-point calibration curves were prepared with concentration ranging from 0.1040µg/mL to 10.1562µg/mL for Ampicillin and 0.0510µg/mL to 6.1552µg/mL for Sulbactam. The peak-area ratio (y) of analytes to internal standards was plotted against the nominal concentration ratio (x) of analyte to internal standard to determine the linearity of each calibration curve. Excellent linearity was achieved with correlation coefficients greater than 0.99 for all validation batches [Figure 3a and 3b].

Figure 3a: Representative calibration curve for Ampicillin

Figure 3b: Representative calibration curve for Sulbactam

The concentrations of calibration standards were back calculated to obtain the accuracy of each calibration point. The ranges of the calibration points’ accuracy were 96.87 – 102.55% for both analytes.

Six samples of LLOQ were processed and then injected along with a ‘Precision and Accuracy’ batch to assess the sensitivity of this method. Precision and accuracy for ampicillin at the LLOQs (0.1040µg/mL) were 2.58% and 96.58%, respectively. Similarly, for sulbactam (LLOQ 0.0510µg/mL), precision and accuracy were 4.66% and 105.08%, respectively. For Ampicillin and Sulbactam, Limits of detection were 0.026µg/mL and 0.013µg/mL and signal to noise ratios were more than 276.51 and 139.18, respectively. These indicate that this method is sensitive enough for a pharmacokinetic study. Moreover, a good signal-to-noise thus obtained indicates that the LLOQ can further be lowered for both analytes or the volume of plasma can be decreased. This further widens the application of this method to the pediatric patients where sample volume is always a challenge.

Precision and accuracy:

Precision and accuracy for intra- and inter-day batches for all analytes were determined by six replicate analyses of QC samples (n=6) at four different concentrations – Lower Limit of Quantification (LLOQ), Low Quality Control (LQC), Middle Quality Control (MQC) and High Quality Control (HQC). The respective concentrations for ampicillin were 0.1059 µg/mL, 0.2862 µg/ml, 3.6141 µg/mL and 7.6897 µg/mL for LLOQ, LQC, MQC and HQC. Results of precision and accuracy were presented in Table 2 (a and b). The intra-day and inter-day precisions for ampicillin were within 3.50% for all analytes. The assay accuracy for ampicillin was: 96.27 –103.08 % of the nominal values. Similarly, for sulbactam precision was less than 9.70% and accuracy was 90.18 - 116.60%. The accuracy of the assay was expressed by [(mean observed concentration) / (spiked concentration)] x 100% and precision was evaluated by relative standard deviation (RSD).

Table 2a: Intra-day and inter-day accuracy and precision for the determination of ampicillin in human plasma

Sample ID	LOQQC (Nominal Conc. 0.1059 µg /ml)			LQC (Nominal Conc 0.2862 µg /ml)			MQC (Nominal Conc 3.6141 µg /ml)			HQC (Nominal Conc 7.6897 µg /ml)
Sample ID	Mean calculated Conc (µg/ml)	Mean accuracy (%)	% CV	Mean calculated Conc(µg/ml)	Mean accuracy (%)	% CV	Mean calculated Conc(µg/ml)	Mean accuracy (%)	% CV	Calculated Conc (µg/ml)	Mean accuracy (%)	% CV
PA - 1	0.1039	98.08	2.98	0.2893	101.07	2.10	3.7071	102.57	2.21	7.7697	101.04	1.99
PA - 2	0.1092	103.08	1.58	0.2928	102.29	1.66	3.6816	101.87	1.98	7.7410	100.67	1.55
PA - 3	0.1020	96.27	2.87	0.2788	97.41	1.90	3.6359	100.60	3.50	7.6820	99.90	2.38
Inter-day	0.1050	99.14	3.82	0.2869	100.26	2.77	3.6749	101.68	2.61	7.7309	100.54	1.94

Table 2b: Intra-day and inter-day accuracy and precision for the determination of sulbactam in human plasma

Sample ID	LOQQC (Nominal Conc. 0.0516 µg /ml)			LQC (Nominal Conc 0.1449 µg /ml)			MQC (Nominal Conc 2.1961 µg /ml)			HQC (Nominal Conc 4.6725 µg /ml)
Sample ID	Mean calculated Conc (µg/ml)	Mean accuracy (%)	% CV	Mean calculated Conc(µg/ml)	Mean accuracy (%)	% CV	Mean calculated Conc(µg/ml)	Mean accuracy (%)	% CV	Calculated Conc (µg/ml)	Mean accuracy (%)	% CV
PA - 1	0.0472	91.47	0.42	0.1453	100.30	2.73	2.2750	103.59	1.76	4.7624	101.92	2.19
PA - 2	0.0527	102.10	1.63	0.1491	102.92	1.56	2.2671	103.23	2.29	4.7138	100.88	1.87
PA - 3	0.0465	90.18	4.86	0.1414	97.61	5.43	2.2620	103.00	8.32	4.5703	97.81	2.85
Inter-day	0.0488	94.58	6.40	0.1453	100.28	4.01	2.2680	103.28	4.77	4.6821	100.21	2.83

Table 3a: Matrix effects of ampicillin in human plasma

Matrix ID	LQC analyte area in absence of matrix	LQC analyte area in presence of matrix	LQC matrix factor for analyte	HQC analyte area in absence of matrix	HQC analyte area in presence of matrix	HQC matrix factor for analyte
PL_1402	66669	65737	98.39	1782230	1799241	102.46
PL_1404	67101	67259	100.67	1774040	1787488	101.79
PL_1447	68066	66558	99.62	1764601	1783122	101.54
PL_1448	63718	67016	100.30	1759473	1796049	102.27
PL_1449	67656	67127	100.47	1741784	1801807	102.60
PL_1450	67665	68187	102.06	1714578	1811244	103.14
LPL_1417		68638	102.73		1779067	101.31
HPL_1321		68604	102.68		1775102	101.08
Average	66812.50	67390.75	100.86	1756117.67	1791640.00	102.02
SD	1593.06	1022.41	1.53	24565.37	12457.2434	0.7094
%CV	2.38	1.52	1.52	1.40	0.70	0.70

Table 3b: Matrix effect for Sulbactam in human plasma

Matrix ID	LQC analyte area in absence of matrix	LQC analyte area in presence of matrix	LQC matrix factor for analyte	HQC analyte area in absence of matrix	HQC analyte area in presence of matrix	HQC matrix factor for analyte
PL_1402	30084	31922	105.67	951259	1004843	104.22
PL_1404	30001	33114	109.61	943062	1022896	106.09
PL_1447	30492	31144	103.09	994347	1036380	107.49
PL_1448	28844	31732	105.04	961189	1030826	106.92
PL_1449	31088	32022	106.00	989545	1028473	106.67
PL_1450	30750	32421	107.32	945427	1024531	106.26
LPL_1417		31025	102.70		1042050	108.08
HPL_1321		31229	103.37		1037334	107.59
Average	30209.83	31826.12	105.35	964138.17	1028416.62	106.67
SD	783.26	710.39	2.35	22480.94	11562.89	1.20
%CV	2.59	2.23	2.23	2.33	1.12	1.12

Anion exchange column was used to nullify the matrix effect. Both ampicillin and sulbactam contain 6-aminopenicillanic acid having a negative charge (Pka of 2.8 and 3.09 respectively). After precipitation with acetonitrile during sample extraction process, 0.1% acetic acid was added to the supernatant to allow both the analytes to retain the negative charge. This enhances the binding of these molecules to the positively charged resin. A mobile phase consisting of ammonium acetate and acetonitrile then used to selectively elute these analytes from the column.

Dilution integrity:

Dilution integrity was evaluated after spiking interference free human plasma with 2 times of HQC concentration of ampicillin and sulbactam. The spiked plasma was diluted either 2 fold (2T) or 4 fold (4T) with interference free human plasma. These samples (Six replicates of each dilution) were processed and then analyzed against a set of freshly spiked calibration standards. The mean accuracy and precision for ampicillin were 111.14% and 0.53% for 2T and 114.48% and 0.75% for 4T. Similarly, for sulbactam the respective values were 107.96% and 0.63% for 2T and 111.90% and 1.81% for 4T. All values were within the acceptance criteria.

Carry – over Effect:

To avoid any carry – over of injected sample in subsequent runs, the cleaning ability of mobile phase used for rinsing the injection needle and port was evaluated. The order of placing samples was: LLOQ of individual analyte, extracted blank plasma, upper limit of quantitation (ULOQ) of individual analyte and extracted blank plasma. No carry – over was observed during the experiment.

Stability:

Stability evaluations were performed in both aqueous and matrix-based samples. For aqueous solution, both short-term and long-term stabilities were determined as follows:

a) Stability in aqueous solution:

i) Short – Term stock solution stability (STSS):

Stock solutions of both analytes and IS were prepared separately and kept at 25°C for 99h and named as stability stock. These stability stock solutions were diluted to provide analytes at LQC and HQC concentrations and intended concentration of IS were prepared and stored at room temperature and named as stability working solution. and stored at 25°C for 98h and marked as stability working solution. Six replicate injections were given for LQC and HQC samples (both stock and working solutions) and diluted IS solutions. Stability stock solutions were diluted to LQC and HQC concentrations just before injections. No significant differences were noticed when these results were compared with those obtained from the freshly prepared LQC and HQC solutions indicating that both analytes were stable at 25°C (Table -4 a and b). Acceptance criteria for the ratio of mean response for stability samples should be between 90-110%.

Table 4a: Short and long –term stability of ampicillin (HQC) aqueous solution

Short-term stability of stock solution at 25°C for 99h

Short-term stability of working solution at 25°C for 98h

Long-term stability of stock solution at

2-8°C for 14 days

Average area of stock solution

Average area of fresh stock solution

% Stability

Average area of working solution

Average area of fresh working solution

% Stability

Average area of stock solution

Average area of fresh stock solution

% Stability

1965897.8333

1974994.1667

99.32

1960855.6667

1974994.1667

99.06

1093402.8333

1099494.8333

99.74

Table 4b: Short and long –term stability of sulbactam (HQC) aqueous solution

Short-term stability of stock solution at 25°C for 99h			Short-term stability of working solution at 25°C for 98h			Long-term stability of stock solution at 2-8°C for 14days
Average area of stock solution	Average area of fresh stock solution	% Stability	Average area of working solution	Average area of fresh working solution	% Stability	Average area of stock solution	Average area of fresh stock solution	% Stability
1265725.8333	1253320.3333	100.97	1274469.8333	1253320.3333	101.66	339923.8333	344124.1667	98.67

ii) Long term stock solution stability (LTSS):

Aqueous LQC and HQC samples of analytes with known concentration of IS were prepared by dilution from respective stock solutions and stored at 2-8°C for 14 days. Mean area responses of LQC and HQC of stored stock solution were then compared against LQC and HQC from freshly prepared stock solution. Similarly, mean area response for internal standard was also compared. Mean percent stabilities were 106.09% (LQC) and 99.74% (HQC) for ampicillin, 105.52% (LQC), 98.67% (HQC) for sulbactam and 101.98% for ampicillin D5 (data not shown) were well within accepted limit (85% - 115%.). This indicated the stability of analytes and internal standard solutions for 14 days at 2-8°C (Table 4 a and b).

b) Stability in human plasma:

i) Bench-top stability:

Six aliquots of each analyte in human plasma (at LQC and HQC concentrations) from the -70⁰C were allowed to thaw unassisted in wet ice bath for 9 h and processed along with a set of freshly prepared calibration standards as well as LQC and HQC samples. The stabilities for LQC and HQC samples of ampicillin were 104.24% and 108.49% respectively. Similarly, for sulbactam, stabilities for LQC and HQC samples were 111.41% and 108.60%. These data were within the acceptance criteria (85-115%).

ii) Freeze thaw stability:

After 4 freeze thaw cycles the stabilities of ampicillin were 100.09% for LQC and 103.06% for HQC, respectively. Similarly, for sulbactam the values were 109.77% and 104.19% respectively. Accepted values are 85 – 115%.

iii) In-injector stability:

The stability for LQC and HQC samples kept in auto-sampler at 10⁰C for 33 h were: 104.32% and 106.15% for ampicillin and 110.93% and 106.48% for sulbactam, respectively.

iv) Wet extract stability:

The stability of ampicillin after 3h of processing at 25°C was 87.61% for LQC and 86.88% for HQC. For sulbactam these values were 91.65% and 89.05% respectively. As per FDA, accepted range for all the stability studies mentioned above is that the mean concentration for stability samples should be 85-115% of the mean concentration of freshly prepared samples. Thus, all the analytes were stable during the analysis process.

Results of stability studies were provided in Table 5 (a and b).

Table 5a: Stability studies of ampicillin in plasma

Parameters	Bench-top stability for 9h			Freeze-thaw stability after 4 cycles		In-injector stability for 33h		Wet extract stability for 3h
Parameters	LQC	HQC	LQC		HQC	LQC	HQC	LQC	HQC
Nominal concentration (µg/ml)	0.2862	7.6897	0.2862		7.6897	0.2862	7.6897	0.2862	7.6897
Mean Calculated concentration (µg/ml) (µg/ml) (ng/ml)	0.3113	8.2425	0.2989		7.8301	0.3115	8.0487	0.2627	6.7695
SD	0.0069	0.2184	0.0061		0.1548	0.0023	0.1701	0.0050	0.0814
%CV	2.20	2.65	2.06		1.98	0.74	2.11	1.89	1.20
% Stability	104.24	108.49	100.09		103.06	104.32	106.15	87.61	86.88

Table 5b: Stability studies of sulbactam in plasma

Parameters	Bench-top stability for 9h		Freeze-thaw stability after 4 cycles		In-injector stability for 33h		Wet extract stability for 3h
Parameters	LQC	HQC	LQC	HQC	LQC	HQC	LQC	HQC
Nominal concentration (µg/ml)	0.1449	4.6725	0.1449	4.6725	0.1449	4.6725	0.1449	4.6725
Mean Calculated concentration (µg/ml)	0.1564	4.9645	0.1541	4.7629	0.1557	4.8676	0.1297	4.1429
SD	0.0030	0.1388	0.0032	0.1110	0.0028	0.0935	0.0023	0.0484
%CV	1.94	2.80	2.09	2.33	1.83	1.92	1.74	1.17
% Stability	111.41	108.60	109.77	104.19	110.93	106.48	91.65	89.05

Table 6a: Extended precision and accuracy of ampicillin

LQC

HQC

Nominal conc.

(µg/ml)

Mean calculated conc. (µg/ml)

Accuracy

(%)

% CV

Nominal conc.

(µg/ml)

Mean calculated conc. (µg/ml)

Accuracy

(%)

% CV

0.2862

0.2834

99.01

2.86

7.6897

7.5668

98.40

2.40

Table 6b: Extended precision and accuracy of sulbactam

LQC

HQC

Nominal conc.

(µg/ml)

Mean calculated conc. (µg/ml)

Accuracy

(%)

% CV

Nominal conc.

(µg/ml)

Mean calculated conc. (µg/ml)

Accuracy

(%)

% CV

0.1449

0.1466

101.19

3.18

4.6725

4.5785

97.99

3.54

CONCLUSION:

This LC–MS/MS method for simultaneous estimation of ampicillin and sulbactam in human plasma is relatively simple, fast, sensitive and specific. Although Nalbant et al described a LCMS/MS method for simulataneous estimation of ampicillin and sulbactam, however, it has used a gradient chromatographic elution to separate analytes and the lower limit of quantification was 0.25 μg/mL for both analytes. On the other hand, a simple isocratic mobile phase was used in our method and lower limit of quantification for sulbactam achieved was 0.13 μg/mL indicating that this method is more sensitive. Moreover, a simple protein precipitation technique which offers consistent and reproducible recoveries with insignificant interference and matrix effect. Moreover, this method does not have any carry – over problem. FDA guideline mentions that internal standard should preferably be identical to the analyte and hence this method was developed using deuterated ampicillin. This method is also validated as per this guideline. By using 100 μL plasma samples, the lower limits of quantification were achieved. It demonstrates that the method is reproducible, sensitive and suitable for high-throughput sample analysis. Moreover, as the sensitivity of this method is quite high, this can be used even for analysis of pediatric samples where sample volume is always a challenge. This method has the potential to be useful for bioequivalence studies and routine therapeutic drug monitoring.

ACKNOWLEDGEMENT:

The authors thank the management of Norwich Clinical Services for providing the opportunity to complete the project.

COMPETING INTERESTS:

All authors hereby declare that no competing of interests is associated with the publication of this manuscript.

AUTHORS' CONTRIBUTIONS:

All authors have equal contribution in this work.

REFERENCES:

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Received on 20.10.2022 Modified on 15.11.2022

Asian J. Pharm. Ana. 2023; 13(1):13-20.

DOI: 10.52711/2231-5675.2023.00003