Simultaneous analysis of Alternaria toxins and citrinin in tomato: an optimised method using liquid chromatography-tandem mass spectrometry

Figures & data

Figure 1. Structure of toxins: logP and pK_a values.

Table 1. Ion source settings on Ultima PT and Thermo TSQ Quantum instruments.

Settings	Ultima PT	Thermos TSQ Quantum
Source temperature (°C)	125	325
Desolvation temperature (°C)	370
Drying gas flow (l h^–1)	902
Cone gas flow (l h^–1)	76
Capillary voltage (kV)	−2.8	−3.0
Collision gas pressure (bar)	2.7 × 10^–6	2.0 × 10^–6
Sheath gas pressure (Arb)		30
Ion sweep gas pressure (Arb)		10
Aux gas pressure (Arb)		5
Capillary temperature (°C)		325°C

Note: Ion mode: ESI; ion polarity: negative; Arb: arbitrary unit.

Table 2. MS/MS ion transitions, settings, and ion ratios on Ultima PT and TSQ Quantum instruments.

Compounds	Detector time segment (min)	Precursor ion [M – H]^– (m/z)	Product ions (m/z)	Cone/tube lens voltage (V)	Collision energies (eV)	Dwell time (s)	Ion ratio % in calibration solutions	Maximum permitted tolerance	Ion ratio % in samples
Ultima PT
ALT	6.5–7.7	291.2	248.1	50	20	0.170	98.1	78.5–117.7	97.0–103.9
			202.9		30	0.170
CIT	6.5–7.7	249.1	205.1	35	15	0.170	40.4	30.3–50.5	39.1–41.7
			177.0		20	0.170
AOH	7.7–8.7	257.1	215.0	50	20	0.125	34.5	25.9–43.1	30.8–38.2
			146.7		20	0.125
TEN	7.7–8.7	413.5	271.2	50	15	0.125	74.2	59.4–89.0	67.6–82.6
			214.8		15	0.125
TEA	7.7–8.7	376.4	301.0	50	15	0.125	89.6	71.7–107.5	75.2–90.1
			329.1		15	0.125
AME	8.7–10	271.1	256.2	50	20	0.250	16.2	11.3–21.1	14.6–20.0
			228.2		20	0.250
TSQ Quantum Ultra
ALT	0–25	291.2	203.2	130	30	0.060	107.2	85.8–128.6	86.6–120.8
			248.1		25	0.060
CIT	0–25	249.2	205.1	120	15	0.060	27.4	20.6–34.3	26.5–30.8
			177.3		20	0.060
AOH	0–25	257.0	215.2	120	25	0.060	40.5	30.4–50.6	32.6–43.9
			147.0		30	0.060
TEN	0–25	413.3	271.2	120	15	0.060	25.4	19.1–31.8	22.4–30.9
			215.1		20	0.060
TEA	0–25	376.2	301.2	80	20	0.060	81.0	64.8–97.2	72.2–97.0
			329.1		15	0.060
AME	0–25	271.0	256.2	100	25	0.060	28.1	21.1–33.7	22.2–30.5
			228.2		30	0.060

Note: Ion mode: ESI; ion polarity: negative; ALT, altenuene; AME, alternariol monomethyl ether; AOH, alternariol; CIT, citrinin; TEA, tenuazonic acid; TEN, tentoxin. Quantifier ions are in bold.

Table 3. Calculated and confirmed analytical limits: validation levels, linearity parameters and matrix effects.

Compound	Calculated LOQ on Ultima PT	Calculated LOQ on TSQ	Estimated LOQ levels set for validation	Validation levels		a	r^2	a	r^2	Absolute ME% and relative ME% (RSD%)	Confirmed LOD on Ultima PT	Confirmed LOQ on Ultima PT	Confirmed LOD on TSQ	Confirmed LOQ on TSQ
	(µg kg^−1)	(µg kg^−1)	(µg kg^−1)	3× LOQestimated	10× LOQestimated	Matrix free		Matrix matched			(µg kg^−1)	(µg kg^−1)	(µg kg^−1)	(µg kg^−1)
ALT	20	50	50	150	500	249	0.9890	140	0.9980	−44 (22%)	10	20	20	50
CIT	5	5	5	15	50	8715	0.9886	15 480	0.9934	78 (10%)	2	5	2	5
AOH	5	5	5	15	50	9221	0.9916	3021	0.9954	−67 (6%)	2	5	2	5
TEN	5	2.5	5	15	50	12 850	0.9920	9373	0.9994	−27 (5%)	2	5	1	2.5
TEA	10	10	10	30	100	4973	0.9635	12 120	0.9557	144 (3%)	10	20	10	20
AME	2	1	2	6	20	88 914	0.9982	45 146	0.9994	−49 (11%)	1	2	0.3	1

Note: a, Slope of calibration; r², determination coefficient; ME%, matrix effect; LOD, limit of detection; LOQ, limit of quantification.

Figure 2. (colour online) (a) Quantifier MRM chromatograms of a spiked tomato sample. Spiking levels are 50 µg kg^–1 for ALT, 5 µg kg^–1 for CIT, 5 µg kg^–1 for AOH, 5 µg kg^–1 for TEN, 10 µg kg^–1 for TEA and 2 µg kg^–1 for AME; (b) quantifier MRM chromatograms of a blank tomato sample. All analyses were performed on an Ultima PT instrument.

Table 4. Recovery, repeatability, intermediate precision, and relative expanded uncertainty.

	Recovery (%)		RSDr (%)		RSDwR (%)		U* (%)
	Levels		Levels		Levels		Levels
Compounds	3× LOQestimated	10× LOQestimated	3× LOQestimated	10× LOQestimated	3× LOQestimated	10× LOQestimated	3× LOQestimated	10× LOQestimated
ALT	90.8	91.1	5.7	5.9	11.5	7.0	25.0	17.0
CIT	94.2	93.4	5.4	3.5	15.9	6.0	21.4	16.2
AOH	89.0	92.6	8.7	5.4	11.0	6.2	29.7	12.6
TEN	90.0	93.1	9.0	4.8	13.3	5.2	28.0	16.0
TEA	90.8	90.4	14.3	5.9	12.7	7.0	19.8	15.5
AME	89.3	89.0	8.0	5.1	9.1	6.5	32.8	14.8

Note: RSDr (%), repeatability relative standard deviation; RSD_wR (%), intermediate precision relative standard deviation; U* (%), relative expanded uncertainty.

Table 5. Detected concentrations in the proficiency test samples and in the standard solution (Z-scores).

Samples	Compounds	Detected concentration (µg kg^−1 in sample, µg l^−1 in standard solution)	Assigned values (µg kg^−1 in sample, µg l^−1 in standard solution)	Z-scores	Evaluation
1	ALT	< LOQ (25 µg kg^−1)	9.48	–	Satisfactory
	AOH	17.2; 17.1; 15.2	13.9	0.8	Satisfactory
	TEN	9.24; 8.81; 8.94	8.29	0.4	Satisfactory
	TEA	54.1; 51.5; 50.2	53.0	−0.1	Satisfactory
	AME	11.2; 11.4; 11.5	11.0	0.1	Satisfactory
2	ALT	< LOD	–	–	Satisfactory
	AOH	6.05; 5.81; 7.04	6.58	−0.2	Satisfactory
	TEN	< LOD	–	–	Satisfactory
	TEA	29.6; 28.2; 26.2	27.0	0.2	Satisfactory
	AME	1.78; 1.53; 1.63	1.56	0.2	Satisfactory
3	ALT	28.9; 30.7; 32.6	30.0	0.1	Satisfactory
	AOH	35.6; 37.8; 39.0	36.3	0.1	Satisfactory
	TEN	32.5; 29.8; 33.0	27.4	0.7	Satisfactory
	TEA	39.2; 37.0; 39.0	39.1	−0.1	Satisfactory
	AME	36.9; 40.3; 39.2	37.3	0.2	Satisfactory
Standard solution	ALT	6.85; 7.23; 8.37	8.73	−0.6	Satisfactory
	AOH	8.84; 8.56; 7.99	10.2	−0.8	Satisfactory
	TEN	11.4; 10.7; 10.7	10.7	0.1	Satisfactory
	TEA	8.03; 8.20; 7.46	11.0	−1.3	Satisfactory
	AME	12.4; 12.0; 10.9	11.4	0.1	Satisfactory