**2.1. Instrumental conditions of UPLC–ESI–MS/MS for the amino acid analysis and the phenolic compound analysis**

The free amino acids and the phenolic compounds are identified in honey. The amino acid analysis method [1, 12, 16] and the phenolic compound analysis method [2, 11, 12] are easy, fast and reliable procedures without sample clean-up and without derivatization steps. The analyses were performed using an UPLC–ESI–MS/MS instrument, consisting of ultra-performance liquid chromatography with a column manager and heater/cooler, binary system manager, sample manager coupled to a triple quadrupole mass spectrometer equipped with electro spray ionization (ESI). The mass spectrometry parameters, confirmation and quantification mass transition (m/z), and their collision energies are listed in **Tables 1** and **2** for amino acids and phenolic compounds, respectively. Separation operations are accomplished using a C18 column, and gradient mobile phase conditions are given in **Tables 3** and **4**, respectively.


**Table 1.** Chromatographic and MRM method parameters for free amino acids using UPLC-ESI-MS/MS [1].



**Table 2.** Chromatographic and MRM method parameters for the analysis of phenolic compounds using UPLC-ESI-MS/MS [2].

Total ion chromatograms (TIC) of each analyte are displayed in **Figures 1** and **2** for amino acids and phenolic compounds, respectively.

#### **2.2. Extraction procedure for amino acid analysis**

**Amino acid Retention time (min) Quantification** 

82 Honey Analysis

**transition (m/z)**

Glycine 0.58 76.00 30, 44, 76 8, 8, 3 Alanine 0.59 90.00 57.1, 71 8, 8 Serine 0.58 106.00 60, 88 9, 10 Proline 0.67 116.10 43.3, 70,1 22, 12 Valine 0.83 118.10 55, 72 18, 10 Threonine 0.61 120.10 56.1, 74, 84, 102.1 15, 10, 12, 9 4-hydroxy Proline 0.61 132.10 68.11, 86.08 14, 12, 8 Leucine 1.67 132.10 69.2, 86 20, 10 Isoleucine 1.54 132.20 69.2, 86,1 20, 9 Asparagine 0.59 133.10 74, 87.13, 115.1 15, 10, 10 Aspartic acid 0.60 134.10 74, 88 14, 10, 8 Lysine 0.58 147.00 84, 115, 130.1 20, 12, 10 Glutamine 0.58 147.10 84.1, 130,1 16, 10 Glutamic acid 0.61 148.10 84, 102.1, 130.2 15, 12, 8 Methionine 1.00 150.20 56.1, 104.1, 133.2 15, 10, 9 Histidine 0.56 156.10 83.1, 93.1, 110.19 22, 20, 15 Phenylalanine 3.41 166.20 77, 91.2,103.1,120 30, 30, 25, 14 Arginine 0.57 175.20 60, 70, 116 15, 20, 15 Tyrosine 1.35 182.16 123.1, 136.1, 165.06 15, 15, 9 Tryptophan 4.27 205.10 91, 118.1, 188.16 35, 25, 10 Cystine 0.58 241.30 74, 120, 152 25, 20, 12

**Table 1.** Chromatographic and MRM method parameters for free amino acids using UPLC-ESI-MS/MS [1].

Pyrogallol 125.01 > 69.10, 79.04, 81.02 20 17, 17, 14 ESI (-) Homogentisic acid 167.03 > 123.03, 122.08, 108.00 10 20, 20, 10 ESI (-) Protocatechuic acid 153.06 > 108.00, 81.01, 91.01 10 20, 25, 20 ESI (-) Gentisic acid 153.05 > 109.04, 108.03, 81.00 10 20, 20, 12 ESI (-) Pyrocatechol 153.06 > 81.01, 108.00, 109.04 8 20, 25, 20 ESI (-) Galantamine 288.10 > 198.00, 213.09, 230.95 20 32, 23, 17 ESI (+)

**Phenolic compounds Quantification > Confirmatory** 

**transition (m/z)**

**Confirmatory transition (m/z)**

**Cone (V) Collision** 

**energies (V)**

**Mode**

**Collision energies (V)**

To prepare 10% (m/v) water honey solutions, 20% methanol solution (v/v) (20 mL), initially acidified with 0.1% formic acid (v/v), is added to 2.0 g honey samples. The resulting mixtures are placed in an ultrasonic bath at 36°C for 10 min to completely mix the


**Table 3.** Chromatographic conditions for free amino acid analysis [1, 12, 16].


**Table 4.** Chromatographic conditions for phenolic compound analysis [2, 11, 12].

extracts of analysed honey samples and subsequently centrifuged at 4000 rpm and 4°C, then the supernatant is filtered through 0.20-μm-pore diameter polytetrafluoroethylene (PTFE) membranes to remove any solid particles, and added to vials and injected into UPLC-ESI-MS/MS [1].

**Column C18 column (1.7 µm 2.1 × 100 mm)** Mobile phase A 0.5% (v/v) acetic acid in ultrapure water

**Table 3.** Chromatographic conditions for free amino acid analysis [1, 12, 16].

**Column C18 column (1.7 µm 2.1 × 100 mm)**

Mobile phase B methanol/water (50:50, v/v) containing 0.1% formic acid

Gradient Time (min) Flow (mL/min) Mobil phase A (%) Mobil phase B (%)

0.00 0.400 99.00 01.00

2.00 0.400 99.00 01.00

8.00 0.400 30.00 70.00

9.00 0.400 99.00 01.00

10.00 0.400 99.00 01.00

Mobile Phase A 0.1% aqueous formic acid

40°C

Mobile phase B 0.5% (v/v) acetic acid in acetonitrile

Gradient Time (min) Flow (mL/min) Mobile phase A

**Table 4.** Chromatographic conditions for phenolic compound analysis [2, 11, 12].

(%)

0.00 0.650 100.00 00.00 1.00 0.650 99.00 01.00 10.00 0.650 70.00 30.00 12.00 0.650 5.00 95.00 13.00 0.650 99.00 01.00 14.00 0.650 100.00 00.00

Mobile phase B

(%)

40°C

Column oven temp.

Column oven temp.

84 Honey Analysis

Injection volume 1 µL

Injection volume 2 µL

**Figure 1.** Total ion chromatograms (TIC) of free amino acids using UPLC-ESI-MS/MS.

86 Honey Analysis Analysis of Amino Acid and Phenolic Content in Honey by UPLC-ESI-MS/MS http://dx.doi.org/10.5772/67317 87

**Figure 1.** Total ion chromatograms (TIC) of free amino acids using UPLC-ESI-MS/MS.

**Figure 2.** Total ion chromatograms (TIC) of phenolic compounds using UPLC-ESI-MS/MS.

#### **2.3. Extraction procedure for phenolic compound analysis**

Honey sample (10 g) is dissolved in ultrapure water (50 mL) and mixed 5 min via vortex. Then ethyl acetate (50 mL) is added into solution flask and the flask is placed on a shaker for 30 min. After then, the flask settles for the phase separation for 180 min. The water phase is extracted two more times with ethyl acetate, and the combined ethyl acetate extract is evaporated under vacuum at 36°C. The residue is redissolved in methanol (5 mL) and filtered from polytetrafluoroethylene (PTFE) membrane 0.20 μm and added to vials, and 2 μL of the solution is injected into UPLC-ESI-MS/MS [2].
