J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website https://www.jocmr.org

Original Article

Volume 16, Number 6, June 2024, pages 293-301

Comparison Between Twenty-Four-Hour Collection and Single Spot Urines for the Detection of Biogenic Amines by High-Performance Liquid Chromatography Tandem Mass Spectrometry

Figure 1. Correlation plots between dopamine (a), 3-methoxytyramine (b), total metanephrine (c), free metanephrine (d), total normetanephrine (e), and free normetanephrine (f) as a ratio to creatinine concentration measured in 24-h collection and spot urine samples.

Figure 2. Correlation plots between epinephrine (a) and norepinephrine (b) as a ratio to creatinine concentration measured in 24-h collection and spot urine samples.

Figure 3. Computed tomography (CT) performed with the administration of an intravenous contrast media showed a well-defined, large and roundish mass (50 × 60 mm) in the right adrenal gland. The neoformation had a weak and uneven enhancement in the dynamic phases (arrow).

**Table 1.** General Clinical Characteristics of the Study Population
Clinical characteristics	Patients, n (%)
PPGL: pheochromocytomas and paragangliomas.
Gender
Male	25 (50%)
Female	25 (50%)
Age, years
18 - 49	16 (32%)
50 - 64	13 (26%)
≥ 65	21 (42%)
Inclusion criteria: clinical suspicion of PPGL
Presence of signs
Hypertension	43 (86%)
Tachycardia	29 (58%)
Sweating	23 (46%)
Hyperglycemia	20 (40%)
Pallor	19 (38%)
Weight loss	3 (6%)
Decreased peristalsis	2 (4%)
Presence of symptoms
Headache	41 (82%)
Anxiety	36 (72%)
Weakness, fatigue	35 (70%)
Palpitations	25 (50%)
Nausea and vomiting	14 (28%)
Exclusion criteria
Simultaneous therapy with drugs that modify catecholamine metabolism	0 (0%)
Not correct sample collection and storage	0 (0%)

Table 1. General Clinical Characteristics of the Study Population

Clinical characteristics

Patients, n (%)

PPGL: pheochromocytomas and paragangliomas.

Gender

Male

25 (50%)

Female

25 (50%)

Age, years

18 - 49

16 (32%)

50 - 64

13 (26%)

≥ 65

21 (42%)

Inclusion criteria: clinical suspicion of PPGL

Presence of signs

Hypertension

43 (86%)

Tachycardia

29 (58%)

Sweating

23 (46%)

Hyperglycemia

20 (40%)

Pallor

19 (38%)

Weight loss

3 (6%)

Decreased peristalsis

2 (4%)

Presence of symptoms

Headache

41 (82%)

Anxiety

36 (72%)

Weakness, fatigue

35 (70%)

Palpitations

25 (50%)

Nausea and vomiting

14 (28%)

Exclusion criteria

Simultaneous therapy with drugs that modify catecholamine metabolism

0 (0%)

Not correct sample collection and storage

0 (0%)

**Table 2.** Binary Gradient Used for HPLC
Time (min)	Phase A	Phase B
HPLC: high-performance liquid chromatography.
0.0	0	100
1.0	0	100
2.0	10	90
2.1	10	90
2.5	30	70
2.6	0	100
3.9	0	100
4.0	0	100

Table 2. Binary Gradient Used for HPLC

Time (min)

Phase A

Phase B

HPLC: high-performance liquid chromatography.

0.0

100

1.0

100

2.0

2.1

2.5

2.6

100

3.9

100

4.0

100

**Table 3.** MRM Transitions and Retention Time Parameters for Catecholamines and Their Metabolites
Analyte	Precursor (Q1) mass (Da)	Fragment (Q3) mass (Da)	XIC width (Da)	Retention time (min)
3-MT: 3-methoxytyramine; DA: dopamine; E: epinephrine; NE: norepinephrine; MN: metanephrine; NMN: normetanephrine; MRM: multiple reaction monitoring; XIC: extracted ion chromatograms.
DA	154	91	0.2	1.51
E	184	166	0.2	1.75
NE	170	152	0.2	2.73
MN	198	165	0.2	0.96
NMN	184	134	0.2	1.37
3-MT	168	91	0.2	0.90
IS DA D4	158	95	0.2	1.49
IS E D6	190	113	0.2	1.74
IS NE D6	176	158	0.2	2.73
IS MN D3	201	183	0.2	0.96
IS NMN D3	187	137	0.2	1.35

Table 3. MRM Transitions and Retention Time Parameters for Catecholamines and Their Metabolites

Analyte

Precursor (Q1) mass (Da)

Fragment (Q3) mass (Da)

XIC width (Da)

Retention time (min)

3-MT: 3-methoxytyramine; DA: dopamine; E: epinephrine; NE: norepinephrine; MN: metanephrine; NMN: normetanephrine; MRM: multiple reaction monitoring; XIC: extracted ion chromatograms.

154

0.2

1.51

184

166

0.2

1.75

170

152

0.2

2.73

198

165

0.2

0.96

NMN

184

134

0.2

1.37

3-MT

168

0.2

0.90

IS DA D4

158

0.2

1.49

IS E D6

190

113

0.2

1.74

IS NE D6

176

158

0.2

2.73

IS MN D3

201

183

0.2

0.96

IS NMN D3

187

137

0.2

1.35

**Table 4.** PCC Obtained for DA, 3-MT, Total MN and Free MN, Total NMN and Free NMN and Type of Correlation Between Data of 24-h and Spot Urine Samples
	DA	3-MT	Total MN	Free MN	Total NMN	Free NMN
PCC: Pearson’s correlation coefficient; 3-MT: 3-methoxytyramine; DA: dopamine; MN: metanephrine; NMN: normetanephrine.
PCC	0.788	0.889	0.904	0.877	0.865	0.777
Correlation	Strong	Strong	Strong	Strong	Strong	Intermediate

Table 4. PCC Obtained for DA, 3-MT, Total MN and Free MN, Total NMN and Free NMN and Type of Correlation Between Data of 24-h and Spot Urine Samples

3-MT

Total MN

Free MN

Total NMN

Free NMN

PCC: Pearson’s correlation coefficient; 3-MT: 3-methoxytyramine; DA: dopamine; MN: metanephrine; NMN: normetanephrine.

PCC

0.788

0.889

0.904

0.877

0.865

0.777

Correlation

Strong

Intermediate

**Table 5.** Biogenic Amines Concentrations (µg/g Creatinine) of the Two Pathological Patients, Obtained Analyzing Both Spot and 24-h Urine Samples
	DA	E	NE	Total MN	Free MN	Total NMN	Free NMN	3-MT
3-MT: 3-methoxytyramine; E: epinephrine; NE: norepinephrine; DA: dopamine; MN: metanephrine; NMN: normetanephrine.
Patient 1
Spot urine	410.0	24.0	10,685.6	380.6	59.7	25,057.5	4,321.8	935.1
24-h urine	502.4	37.6	10,071.4	228.9	55.2	24,878.5	3,909.5	900.7
Patient 2
Spot urine	317.7	71.4	360.3	3,454.7	526.0	4,917.7	998.3	248.9
24-h urine	208.4	92.7	301.8	4,374.2	583.4	4619.0	856.0	298.0

Table 5. Biogenic Amines Concentrations (µg/g Creatinine) of the Two Pathological Patients, Obtained Analyzing Both Spot and 24-h Urine Samples

Total MN

Free MN

Total NMN

Free NMN

3-MT

3-MT: 3-methoxytyramine; E: epinephrine; NE: norepinephrine; DA: dopamine; MN: metanephrine; NMN: normetanephrine.

Patient 1

Spot urine

410.0

24.0

10,685.6

380.6

59.7

25,057.5

4,321.8

935.1

24-h urine

502.4

37.6

10,071.4

228.9

55.2

24,878.5

3,909.5

900.7

Patient 2

Spot urine

317.7

71.4

360.3

3,454.7

526.0

4,917.7

998.3

248.9

24-h urine

208.4

92.7

301.8

4,374.2

583.4

4619.0

856.0

298.0