Validation and application of a simple and rapid stability-indicating liquid chromatographic assay for the quantification of caffeine from human saliva

Figures & data

Table 1. Accuracy, precision, and extraction recovery data for caffeine citrate at concentrations of 1, 15, and 30 µg/mL.

Concentration (µg/mL)	Intra-day (n = 3)		Inter-day (n = 9)		Extraction recovery (n = 9)
	Accuracy (% Nominal)	Precision (% RSD)	Accuracy (% Nominal)	Precision (% RSD)	Mean (%)	RSD (%)
1	97.8	3.1	94.7	4.0	110.6	3.5
15	98.8	0.9	96.9	6.9	95.0	6.8
30	97.7	1.4	99.7	5.2	95.1	5.2

RSD: relative standard deviation.

All values are expressed as a mean percent (%).

Figure 1. Stacked chromatograms of blank and spiked saliva samples (15 µg/mL) and calibration standard (15 µg/mL) at 273 nm, with caffeine peak eluting at 6.35 min. Graphs in the dotted box show caffeine spectra and representative peak purity of the samples.

Table 2. Stability of caffeine in saliva under refrigeration (2–8 °C), at room temperature (20–25 °C), and frozen (−20 °C).

Concentration (µg/mL)	Percentage of caffeine remaining
	24 hr			48 hr			7 days
	20–25 °C	2–8 °C	−20 °C	20–25 °C	2–8 °C	−20 °C	20–25 °C	2–8 °C	−20 °C
0.5	96.8 (13.3)	98.0 (12.0)	76.3 (20.0)	100.5 (18.7)	106.7 (32.2)	80.1 (7.5)	107.1 (19.9)	97.1 (30.7)	72.5 (1.2)
3	97.0 (2.9)	104.7 (1.42)	92.1 (11.9)	101.1 (3.1)	100.3 (2.7)	95.3 (6.6)	97.2 (1.74)	101.2 (1.24)	106.9 (7.8)

Data presented as mean (SD); n = 3.

Table 3. Stability evaluation of extracted caffeine samples from saliva kept in auto sampler for 72 hr and for three freeze-and-thaw cycles (from −20 °C to room temperature) for n = 3 samples each.

Concentration (µg/mL)	Auto sampler, 48 hr		Three cycles, freeze-and-thaw
	Drug remaining (%)	RSD (%)	Drug remaining (%)	RSD (%)
1	106.7	1.2	102.0	5.4
15	103.5	2.7	92.6	7.8
30	103.7	2.9	102.0	8.5

RSD: relative standard deviation.

Data are mean values.

Table 4. pH of caffeine citrate samples [mean (SD); n = 3] stored at room temperature for up to 3 months.

Timepoint	pH of solution
	Concentration (mg/mL)
	5	10	15	20
Baseline	2.43 (0.16)	2.15 (0.01)	2.06 (0.03)	1.90 (0.06)
7 days	2.53 (0.02)	2.29 (0.03)	2.20 (0.02)	2.16 (0.02)
14 days	2.44 (0.05)	2.22 (0.08)	2.13 (0.03)	2.03 (0.02)
21 days	2.46 (0.03)	2.25 (0.00)	2.15 (0.01)	2.07 (0.05)
28 days	2.49 (0.05)	2.33 (0.02)	2.23 (0.02)	2.09 (0.02)
6 weeks	2.44 (0.02)	2.21 (0.04)	2.09 (0.01)	1.95 (0.05)
2 months	2.52 (0.04)	2.35 (0.01)	2.20 (0.03)	2.16 (0.01)
3 months	2.31 (0.03)	2.09 (0.01)	1.91 (0.02)	1.85 (0.04)

Figure 2. Concentration of 5 (●), 10 (▪), 15 (▲), and 20 (▼) mg/mL caffeine citrate solutions over a three-month period. Data presented as mean ± SD; n = 3.