Combining LC–MS/MS and Hollow-Fiber Infection Model For Real-Time Quantitation of Ampicillin to Antimicrobial Resistance

Figures & data

Figure 1.  Chemical structures of ampicillin and ampicillin-d5 (IS).

Figure 2.  Representative chromatograms of ampicillin and ampicillin-d5 in blank Luria–Bertani broth, lower limit of quantification sample and upper limit of quantification sample.

LLOQ: Lower limit of quantification; ULOQ: Upper limit of quantification.

Figure 3.  Representative standard curve of ampicillin in Luria–Bertani broth.

Table 1.  Intra- and inter-batch precision and accuracy for ampicillin.

Quality control conc. (μg/ml)	LLOQ (0.10)	LQC (0.30)	MQC (8.00)	HQC (40.00)
Intra-assay precision and accuracy (n = 6 at each QC level)
Mean	0.09	0.32	7.65	38.82
SD	0.01	0.01	0.22	0.37
% CV	11	3	3	1
% Accuracy	90	105	96	97
Inter-assay precision and accuracy (n = 18 at each QC level)
Mean	0.09	0.31	7.49	37.84
SD	0.01	0.02	0.42	1.35
% CV	11	6	6	4
% Accuracy	91	103	94	95

HQC: High quality control; LLOQ: Lower limit of quantification; LQC: Low quality control, MQC: Medium quality control.

Table 2.  Extraction recovery and matrix effect of ampicillin in Luria&Bertani medium. n = 6 at each QC level.

AMPI conc. (μg/ml)	LQC (0.30)	MQC (8.00)	HQC (40.00)
Mean percent extraction recovery	105	100	99
Mean percent extraction recovery (AMPI-d5)	100
Matrix factor (IS normalized)	1.1	1.0	1.1

AMPI: Ampicillin; High-quality control; LQC: Low-quality control, MQC: Medium-quality control.

Table 3.  Stability data for ampicillin in Luria–Bertani medium. 

Stability test	Spiked QC conc. (μg/ml)	Mean ± SD (μg/ml)	%CV (precision)	% Stability (accuracy)
Autosampler^†	0.30	0.31 ± 0.03	10	105
	40.00	37.98 ± 1.54	1	95
Freeze–thaw^‡	0.30	0.33 ± 0.02	6	109
	40.00	38.72 ± 0.46	1	97
Benchtop^§	0.30	0.32 ± 0.02	6	108
	40.00	38.54 ± 0.51	1	96

† Up to 72 h in autosampler at 4°C.

‡ Up to 3 freeze–thaw cycles at -20°C.

§ At room temperature for up to 12 h.

n = 6 at each QC level.

CV: Coefficient variant; QC: Quality control; SD: Standard deviation.

Figure 4.  Correlation of in vitro experimental versus simulated PK data for ampicillin in Luria&Bertani broth.