Identification of Phytochemicals of Phoenix dactylifera L. Cv Ajwa with UHPLC-ESI-QTOF-MS/MS

ABSTRACT

Date palm (Phoenix dactylifera) fruit is an important food in the Middle East. Some date palm cultivars like the Ajwa is revered for its health-promoting properties. Phytochemicals like phenolic compounds are in part responsible for such health benefits. However, detailed information on these compounds is lacking. The present work aimed to investigate phytochemical components of Ajwa cultivar using UHPLC-ESI-QTOF-MS/MS in three different extraction solvents (aqueous, methanol–water, acetone–water) and three different extraction durations (2, 5 and 24 h). The proposed method provided tentative identification of 169 bioactive compounds out of which 44 (polyphenols and other phytochemical compounds) were successfully identified from three different extracts and three different extraction durations. Twenty-one compounds never previously reported in the Ajwa cultivar were identified. Aqueous-based extraction solvent and 24-h extraction duration yielded most phytochemical compounds.

KEYWORDS:

• Ajwa cultivar
• Phoenix dactylifera
• UHPLC-ESI-QTOF-MS/MS
• extraction solvent

Introduction

Date palm, Phoenix dactylifera from Arecaceae family, is an important food crop in the Middle East. It is primarily cultivated for its sweet edible fruit which can be eaten fresh or processed into various products like vinegar, syrup, drink, powder and confectionery (Al-Shahib and Marshall, 2003; Salah et al., 2010). Other parts of the date palm like leaves and timber may also be useful as traditional building material (Riahi et al., 2009; Zabar and Borowy, 2012). More than 2000 cultivars have been developed over centuries from distinctive regions across the Middle East and the Mediterranean. The Ajwa cultivar, developed and cultivated only in Madinah, Saudi Arabia is one of the most expensive and highly revered cultivar in the market mostly due to religious and ethnomedical belief of it as functional food for health (Yasin et al., 2015). Evidence is emerging for the cardioprotective effects of Ajwa date (Al-Yahya et al., 2016; Khan and Siddiqui, 2017). Anticancer effects of Ajwa date have been demonstrated in rodent models (Khan et al., 2018) and cancer cell lines for breast (Khan et al., 2016), prostate (Mirza et al., 2018) and colon (Eid et al., 2014) cancers. Other effects include liver (Sheikh et al., 2014), gut (Eid et al., 2014), tissue (Ragab et al., 2013) and kidney (Ali et al., 2011) protection. Ajwa date is also effective against pathogens like fungi (Belmir et al., 2016). However, few investigators studied nutritional and phytochemical contents of Ajwa date. Assirey (2015) studied nutritional composition of different date cultivars and demonstrated that Ajwa date contained the highest amount of minerals compared to other cultivars like Mabroom, Shalaby, Safawy, Khodari, Brni, Anabarah, Suqaey, Sukkari and Labanah. Eid et al. (2013) demonstrated the superiority of Ajwa cultivar compared to Barni and Khalas cultivar in terms of phenolic content using liquid chromatography mass spectrometry analysis on methanol extract. Zhang et al. (2013) confirmed the bioactivity of Ajwa date using antioxidant and anti-inflammatory assays. However, little is known about the phytochemical components of Ajwa date responsible for its therapeutic effects. Thus, detailed and extended profiling of the phytochemicals of Ajwa date using high sensitive tools is necessary. Mass spectrometry coupled to high-performance liquid chromatography (HPLC–MS/MS) has been increasingly used in the structural characterization of complex matrices and has been proven to be the tool of choice to identify phenolic compounds (Abu-Reidah et al., 2015). Different extraction techniques (Wang and Weller, 2006), solvents (González-Montelongo et al., 2010) and duration (Chew et al., 2011) may influence the amount and type of biochemical component yield. Using a potent analytical technique like ultra high-performance liquid chromatography electrospray time of light mass spectrometry (UHPLC-ESI-QTOF-MS/MS) we investigated the phytochemical composition of Ajwa date on different extraction solvents and duration.

Materials and Methods

Chemicals

Methanol (CH₄O) of analytical grade was purchased from John Kollin Corporation (Midlothian, UK). Acetone (C₃H₆O) of analytical grade was purchased from R&M Chemicals (Semenyih, Malaysia) 0.1 formic acid, 5 mM ammonium formate and acetonitrile were of UHPLC-QTOF-MS/MS grade.

Sample Preparation

Ajwa dates (500 g) were purchased from a local supplier Syarikat Abdul Ghaffar Sdn. Bhd. (Penang, Malaysia) who imports from Madinah, Saudi Arabia. The flesh was washed and dried at room temperature for 24 h and then kept at room temperature until further use.

Extraction

Aqueous Extract

Aqueous extract was prepared according to the protocols of Vayalil (2002) and El-Arem et al. (2014) with slight modifications. 40 g of Ajwa date flesh were ground with pestle and mortar and soaked in 120 mL distilled water then left at room temperature for either 2, 5 or 24 h. The mixtures were filtered using sterile cotton gauze followed by Whatman® no. 1 filter paper (Whatman Int. Ltd., Maidstone, UK) and the filtrates collected and kept at −80°C until further use.

Methanol–water Extract (80%)

Methanol–water extract was prepared according to the protocols of Singh et al. (2012) and Odeh et al. (2014) with slight modification. Forty grams of Ajwa date flesh were ground with mortar and pestle and dissolved in 120 mL methanol–water (80%), then left at room temperature for either 2, 5 or 24 h. The solutions were filtered using sterile cotton gauze followed by Whatman® no.1 filter paper (Whatman Int. Ltd., Maidstone, UK). The filtrates were collected then concentrated using IKA® RV10 rotary evaporator (IKA-Werke GmbH, Staufen, Germany) and JEIO TECH VE-11 electrical aspirator (JEIO TECH Co. Ltd., Seoul, Korea) to approximately 20 mL. The extracts were kept at −80°C until further use.

Acetone–water Extract (70%)

Acetone–water extract was prepared according to the protocols of Williams et al. (2014) and Aïssa et al. (2015) with slight modification. 10 g of Ajwa date flesh were ground with pestle and mortar and dissolved in 100 mL of acetone–water (70%). The solution was left at room temperature for either 2, 5 or 24 h. The solutions were filtered using sterile cotton gauze followed by Whatman no.1 filter paper (Whatman Int. Ltd., Maidstone, UK). The filtrates were collected then concentrated using IKA® RV10 rotary evaporator (IKA-Werke GmbH, Staufen, Germany) and JEIO TECH VE-11 electrical aspirator (JEIO TECH Co. Ltd., Seoul, Korea) to approximately 20 mL. The extracts were kept at −80̊C until further used for analysis.

UHPLC-ESI-QTOF-MS/MS Analysis

All the extracts were subjected to UHPLC-ESI-QTOF-MS/MS analysis conducted by Advanced Chemistry Solutions Sdn. Bhd., Petaling Jaya, Malaysia. The analysis was carried out on the Eksigent® 110 Ultra-High-Performance Liquid Chromatography system (AB Sciex Pte. Ltd., Biopolis, Singapore) coupled with Sciex TripleTOF® 5600 + high resolution accurate mass tandem mass spectrometry (AB Sciex Pte. Ltd., Biopolis, Singapore). All samples were filtered with Merck Millex® 0.45-µm nylon syringe filter (Merck & Co., Kenilworth, New Jersey, USA) before injection into LC-MS/MS. 10 µL of sample was injected via the Eksigent® 110 UHPLC autosampler (AB Sciex Pte. Ltd., Biopolis, Singapore) and compound separation achieved with a reverse phase Phenomenex® Synergy RP C-18 column (100 × 2.0 mm × 3 µm) (Phenomenex Inc., California, USA). Sample elution was carried out using gradient elution comprising solvent A [water: 0.1% formic acid (Merck & Co., Kenilworth, New Jersey, USA): 5 mM ammonium formate (Merck & Co., Kenilworth, New Jersey, USA)] and solvent B [acetonitrile (Merck & Co., Kenilworth, New Jersey, USA): 0.1% formic acid (Merck & Co., Kenilworth, New Jersey, USA): 5 mM ammonium formate (Merck & Co., Kenilworth, New Jersey, USA)]. The following gradient was used for the chromatography separation: 5% B to 95% B from 0.01 to 20 min, hold for 5 min and back to 5% B in 0.1 min and re-equilibrated for 5 min. The Sciex TripleTOF ® 5600 + high resolution accurate mass spectrometer (AB Sciex Pte. Ltd., Biopolis, Singapore) was used to obtain the mass spectrum. The ionization source used was Sciex patented Duo Spray source with electrospray ionization (ESI) in negative mode. Ionization voltage used was 5500kV with 500°C source temperature. For the identification of phytochemical compounds, the mass scan range was set from m/z 100 to 1200 with information-dependent acquisition (IDA) of mass fragment spectrum. The mass fragmentation spectrum is an important requirement to ensure identification of the compounds. Further identification was done via chemical formula finding based on high resolution accurate mass data obtained. Cross-referencing with existing publications as well SCIEX’s natural products database.

Result and Discussion

In the present work, bioactive composition from the aqueous-based extracts (aqueous, methanol–water and acetone–water) of Ajwa date palm fruit was carried out using UHPLC-ESI-QTOF-MS/MS in negative ionization mode. The method tentatively detected 169 phytochemical compounds, of which 44 were successfully characterized (Table 1). In addition, 21 new compounds were detected for the first time in Ajwa date palm fruit (sinapic acid hexoside, p-coumaroyl hexose, p-hydroxybenzoylhexose, protocatechuic acid hexoside, chrysoeriol, chrysoeriol hexosyl sulfate, luteolin hexoside, luteolin rhamnosyl hexoside, luteolin rhamnosyl dihexoside, dihydrokaempferol hexoside, isorhamnetin rhamnosyl hexoside, isorhamnoetin-3-o-glucoside, chrysoeriol rhamnosyl hexoside, kaempferol-3-o-glucoside-7-o-rhamnoside, afzelin-o-gallate, eriodictyol xyloyl deoxy hexose, pinene-ol-o-glucoside, trihydroxy-9,14-octadecadienoic acid, trihydroxy-octadecenoic acid, hydroxy octadecadienoic acid and gluconic acid). The compounds detected in this work were tentatively characterized by means of MS data, together with the interpretation of the observed MS/MS spectra in comparison with those found in the literature and internal database.

Table 1. Classification of characterized phytochemical compounds in Ajwa date fruit

Classification			Phytochemical compounds	References
Polyphenols	Phenolic acid	Hydroxycinnaminic acids	Caffeic acid	Ahmed et al., 2016a; Hamad et al., 2015; Yasin et al., 2015; Ragab et al., 2013; Saleh et al., 2011.
			Caffeic acid hexoside	Yasin et al., 2015
			Caffeic acid hexoside conjugate	-
			Caffeoyl acid	-
			O-caffeoyl shikimic acid	Farag et al., 2014; Yasin et al., 2015
			O-dicaffeoyl shikimic acid	Farag et al., 2014; Yasin et al., 2015
			O-dicaffeoyl shikimic acid acetate	-
			Sinapic acid hexoside	Farag et al., 2014
			Ferulic acid	Ahmed et al., 2016a; Hamad et al., 2015; Yasin et al., 2015; Martins et al., 2011
			Chlorogenic acid	Ahmed et al., 2016a; Hamad et al., 2015; Reis et al., 2012
			p-Coumaroyl hexose	Amessis-Ouchemoukh et al., 2014
		Hydroxybenzoic acids	p-Hydroxybenzoylhexose	-
			Protocatechuic acid hexoside	Yasir et al., 2016
			Gallic acid	Ahmed et al., 2016a; Hamad et al., 2015; Yasin et al., 2015;
		Flavonoids	Flavones	Apigenin pentosyl hexoside	Yasin et al., 2015
			Apigenin 7-O-neohesperidoside	Yasin et al., 2015
			Chrysoeriol	Farag et al., 2014
			Chrysoeriol hexosyl sulfate	Farag et al., 2014
			Luteolin	Ahmed et al., 2016a; Hamad et al., 2015; Yasin et al., 2015; Farag et al., 2014
			Luteolin hexoside	Farag et al., 2014
			Luteolin rhamnosyl hexoside	Farag et al., 2014
			Luteolin rhamnosyl dihexoside	Farag et al., 2014
		Flavonoles	Quercetin	Ahmed et al., 2016a; Hamad et al., 2015; Yasin et al., 2015; Farag et al., 2014
			Quercetin rhamnosyl dihexoside	Yasin et al., 2015; Farag et al., 2014
			Quercetin 3-hexose	Yasin et al., 2015
			Quercetin 7-O-hexoside-3-O-rhamnoside	Yasin et al., 2015
			Isoquercetin	Ahmed et al., 2016a; Hamad et al., 2015; Yasin et al., 2015; Farag et al., 2014
			Isoquercetrin acetate	Farag et al., 2014; Yasin et al., 2015
			Isoquercetin sulfate	Farag et al., 2014; Yasin et al., 2015
			Dihydrokaempferol hexoside	-
			Rutin	Ahmed et al., 2016a; Hamad et al., 2015; Farag et al., 2014; Martins et al., 2011
			Isorhamnetin rhamnosyl hexoside	Farag et al., 2014
			Isorhamnoetin-3-O-glucoside	Farag et al., 2014
			Chrysoeriol rhamnosyl hexoside	Farag et al., 2014
			Kaempferol-3-O-glucoside-7-O-rhamnoside	-
			Afzelin-O-gallate	-
		Flavanones	Eriodictyol xyloyl deoxy hexose	-
Terpenoids			Pinenol-O-glucoside	-
Fatty acids			Trihydroxy-9,14-octadecadienoic acid	Farag et al., 2014
			Trihydroxy-octadecenoic acid	Farag et al., 2014
			Hydroxy octadecadienoic acid	Farag et al., 2014
Organic acids			Malic acid	Hamad et al., 2015; Abu-Reidah et al., 2015
			Gluconic acid	Ramachandran et al., 2006
			Ascorbic acid	Ahmed et al., 2016a; Yasir et al., 2016

Organic Acids

Malic acid, gluconic acid and ascorbic acid were detected. Malic and ascorbic acids have been previously reported (Hamad et al., 2015).

Phenolic Acids and Derivatives

In the present work, 14 phenolic acid derivatives were identified, and 11 of which were hydroxycinnaminic acids and 3 were hydroxybenzoic acids. The 11 hydroxycinnaminic acids were caffeic acid, caffeic acid hexoside, caffeic acid hexoside conjugate, caffeoyl acid, O-caffeoyl shikimic acid, O-dicaffeoyl shikimic acid, O-dicaffeoyl shikimic acid acetate, sinapic acid hexoside, ferulic acid, chlorogenic acid and p-coumaroyl hexose. The three hydroxybenzoic acids were p-hydroxybenzoylhexose, protocatechuic acid hexoside and gallic acid. Caffeic acid, gallic acid, ferulic acid and chlorogenic acid have already been reported by Saleh et al. (2011); Hamad et al. (2015); Ahmed et al. (2016a).

Flavonoid Derivatives

A total of 24 flavonoid derivatives were detected. Out of this eight were flavones which included apigenin pentosyl hexoside, apigenin 7-O-neohesperidoside, chrysoeriol, chrysoeriol hexosyl sufate, luteolin, luteolin hexoside, luteolin rhamnosyl hexoside, luteolin rhamnosyl dihexoside. 15 were flavonols which included quercetin, quercetin rhamnosyl dihexoside, quercetin 3-hexose, quercetin 7-O-hexoside-3-O-rhamnoside, isoquercetin, isoquercetin acetate, isoquercetin sulfate, dihydrokaempferol hexoside, rutin, isorhamnetin rhamnosyl hexoside, isorhamnetin-3-O-glucoside, chrysoeriol rhamnosyl hexoside, kaempferol-3-O-glucoside-7-O-rhamnoside and afzelin-O-gallate. Only 1 flavanone which included eriodictyol xylol deoxy hexose. Quercetin, luteolin, rutin, iso-quercetin and apigenin have all been reported previously (Ahmed et al., 2016a; Hamad et al., 2015; Saleh et al., 2011).

Terpenoids

For the first time, one terpenoid was detected as pinenol-O-glucoside.

Fatty Acids

For the first time, three fatty acids were detected in Ajwa date palm fruit which include trihydroxy-9,14-octadecadienoic acid, trihydroxy-octadecenoic acid and hydroxy octadecadienoic acid.

UHPLC-ESI-QTOF-MS/MS

The present study used UHPLC-ESI-QTOF-MS/MS, the combination of liquid chromatography and mass spectroscopy is a highly sensitive and accurate technique compared to HPLC alone. The ionization method (electrospray ionization) which was used in the present study has the ability to produce multiply charged molecular ions in comparison to other methods like MALDI (Matrix assisted laser desorption) which leads to formation of single charged molecular ions. Additionally, the present study also used triple quadrupole time-of-flight tandem mass 10 spectrometry (QTOF-MS/MS) to analyze individual component and this technique adds specificity to the given analysis. Date fruit extract was initially analyzed in both negative and positive ion modes (Farag et al., 2014). Our study utilized negative ion mode [M-H]- due to better sensitivity and more observable peaks especially for polyphenolic compounds (Farag et al., 2014). The identification of phytochemical compounds was based on five different parameters: retention time, molecular weight of each compound, m/z [M-H]-, molecular formula and fragmentation pattern. Representative chromatograms of LC-MS/MS data from two extraction samples of Ajwa date fruit in the present study were shown in Figure 1. The chromatograms showed identification of O-dicaffeoyl shikimic acid (MS peak at m/z 497.1) which had retention time at 4.671 min and caffeic acid (MS peak at m/z 179) which had retention time at 4.178 min.

Figure 1. Examples LC-MS/MS data of Ajwa date fruit extract. Ai and Aii: Peak with retention time 4.671 min was analyzed with a pseudomolecular peak [M-H]- at m/z = 497.1 indicating the presence of O-dicaffeoyl shikimic acid and confirmed by MS/MS fragments at m/z = (135.0274, 161.0038, 179.0126, 335.0464). Bi and Bii: Peak with retention time 4.178 min was analyzed with a pseudomolecular peak [M-H]- at m/z 179.0 indicating the presence of caffeic acid and confirmed by MS/MS fragments at m/z = (59.0165, 71.0150, 89.0254, 134.0367, 135.0450, 136.0486)

Effects of Different Extraction Solvents on Identified Compounds

In all the three extracts, extraction duration of 24 h qualitatively yielded most compounds. Although all three solvents were aqueous based, pure aqueous extract yielded more bioactive compounds which include phenolic acids and flavonoids compared to methanol–water and acetone–water-based extraction. This finding is in agreement with other study which has demonstrated that water is a good solvent for extracting phenolic compounds from apple pomace (Reis et al., 2012). Similarly, other studies have also found that the yield of phenolic compounds from aqueous extract was superior than pure organic solvent extract (Do et al., 2014). This indicated that water is the best solvent to extract phenolic acids and flavonoids compared to methanol–water and acetone–water extracts. Besides glycated compounds, sulfated compounds were detected. Though not usually found in natural products, it has been reported in dates (Farag et al., 2014). In the present study, 11 of bioactive compounds were successfully characterized in water extract which included apigenin pentosyl hexoside, gluconic acid, malic acid, afzelin-o-gallate, protocatechuic acid hexoside, p-hydroxy benzoyl hexose, dihydrokaempferol hexoside, isoquercetrin acetate, isorhamnetin rhamnosyl hexoside, eriodictyol xyloyl deoxy hexose and p-coumaroyl hexose (Table 2). Whereas, 10 bioactive compounds were successfully characterized in methanol–water (80%) including luteolin, sinapic acid hexoside, caffeoyl acid, ferulic acid, gallic acid, quercetin, quercetin-7-O-hexoside-3-O-rhamnoside, quercetin-3-hexose, apigenin-7-O-neahesperidoside and caffeic acid hexoside conjucate (Table 3) as well as 8 bioactive compounds appeared in acetone–water (70%) which included ascorbic acid, caffeic acid, kaempferol-3-O-glucoside-7-O-rhamnoside, chlorogenic acid, hydroxy actadecadienoic acid, pinenol-O-glucoside, O-dicaffeoyl shikimic acid and luteolin rhamnosyl dihexoside (Table 4). On the other hand, nine compounds were identified in all three solvents like O-dicaffeoyl shikimic acid, isoquercetin sulfate, isoquercetin, trihydroxy octadecenoic acid, chrysoeriol, O-caffeoyl shikimic acid, chrysoeriol rhamnosyl hexoside, chrysoeriol hexosyl sulfate and luteolin hexoside (Tables 2,3 and 4). Whereas three compounds such as luteolin rhamnosyl dihexoside, trihydroxy-9,14-octadecadienoic acid and rutin appeared only in each of aqueous and acetone–water extracts (Tables 2 and 4), only one compound (caffeic acid hexoside) was detected in each aqueous and methanol–water extracts (Tables 2 and 3) and two compounds like isorhamnetin-3-O-glucoside and quercetin rhamnosyl dihexoside were found only in each of methanol–water and acetone–water extracts (Tables 3 and 4). The proposed method in this study also showed that methanol–water extract has more phytochemical compounds compared to acetone–water extract. Solvent such as methanol has a superior extraction power compared to acetone or ethyl acetate when they are used to extract polar compounds from several medicinal plants (Miliauskas et al., 2004). Polarity of extraction solvent may play an important role in extracting polar compounds. In terms of polarity, methanol is more polar than acetone (Sadek, 2002). Besides that, the yield of extraction also depends on pH, temperature, extraction time and composition of the sample (Do et al., 2014). The extraction yield decreased when using absolute organic solvents.

Table 2. Phytochemical compounds detected after 2, 5 and 24 h of extracted Ajwa date fruit with water using LC/ESI-QTOF-MS/MS

Extraction time	Peak no.	Retention time (min)	Molecular weight	[M-H] m/z	Identified compounds	Molecular formula	Major fragments (MS/MS)	References
2 h	1	2.143	564	563.2	Apigenin pentosyl hexoside	C26H27O14	176.953, 176.937, 285.044, 220.924, 337.061, 393.026, 427.089	Farag et al., 2014
	2	2.467	342	341.1	Caffeic acid hexoside	C15H18O9	59, 71, 73, 89, 101, 113, 119, 131, 143, 161, 179, 207, 221, 251	-
	3	2.496	623	622.1	Unknown	-	292, 310, 343, 416, 460, 532	-
	4	2.573	426	425.2	Unknown	-	73.02, 129.06, 129.02, 128.02, 176.03, 219.04, 218.04, 317.08, 334.08, 335.08, 335.1, 425.04, 425.15, 425.13	-
	5	2.817	196	195.0	Gluconic acid	C6H12O7	59, 71, 74.9, 85, 98.9, 129, 159, 177	-
	6	2.869	237	236	Unknown	-	71.9, 103, 144.1, 146, 147, 164.1, 181, 206.1	-
	7	4.158	300	299.1	p-Hydroxybenzoylhexose	C13H16O8	93, 135.9, 137, 179, 239	-
	8	4.709	326	325.1	p-Coumaroyl hexose	C15H17O8	96.6, 119, 163.1	Amessis-Ouchemoukh et al. (2014)
	9	4.745	498	497.1	O-dicaffeoyl shikimic acid	C22H25O13	135.1, 161, 179, 335	Farag et al., 2014
	10	5.617	450	449.2	Dihydrokaempferol hexoside	C21H22O11	59, 119, 207, 209.1, 251.1, 269, 287, 448.1, 449	Abu-Reidah et al., 2015
	11	5.667	756	755.4	Luteolin rhamnosyl dihexoside	C33H39O20	128, 233, 306, 385.1, 413.1, 531.1, 593.16, 593.1, 593.2, 663.1, 709.18, 709.16, 755.16, 755.2, 755.10, 755.3	Farag et al., 2014
	12	5.742	544	543.0	Isoquercetin sulfate	C21H19O15	150.6, 24.9, 300.9, 301, 463	Farag et al., 2014
	13	6.062	464	463.1	Isoquercetin	C21H19O12	178.9, 255, 270.9, 299.9, 301	Farag et al., 2014
	14	6.378	644	643.2	Unknown	-	165, 191, 284, 299, 342.9, 607.2, 607.1	-
	15	6.471	776	775.4	Unknown	-	150, 165, 195.04, 195.07, 387.11, 387.14, 417.1, 535.1, 565.2, 595.2, 613.2, 775.1, 775.2, 775.3	-
	16	7.475	328	327.2	Trihydroxy-9,14-octadecadienoic acid	C18H31O5	171.1, 183.1, 211.1, 229.1, 309.2	Farag et al., 2014
	17	7.934	330	329.2	Unknown	-	171.1, 211.1	-
	18	8.064	300	299.0	Chrysoeriol	C16H11O6	256, 284	Farag et al., 2014
	19	8.900	294	293.2	Unknown	-	131.1, 177.1, 205.1, 221.1, 220.1, 236.07, 236.1	-
	20	9.468	226	225.0	Unknown	-	59, 71, 89, 113, 119, 127.1, 179, 224.77	-
	21	11.482	340	339.2	Unknown	-	119, 169.9, 182.9, 197, 339.2	-
	22	11.714	180	179.0	Unknown	-	59, 65, 71, 89	-
	23	19.147	689	688.0	Unknown	-	255.2, 363.5, 387.2, 405.2, 387.2, 641.6, 641.5, 687.5	-
	24	20.603	911	910.0	Unknown	-	701.4, 773.5	-
	25	21.009	199	198.0	Unknown	-	160.86, 160.82, 162.6, 197.78	-
	26	22.710	914	913.0	Unknown	-	703.5, 774.5, 774.6, 775.5, 776.5, 912.5, 912.4	-
5 h	1	2.567	326	325.1	p-Coumaroyl hexose	C15H17O8	101, 127, 145, 144	(Amessis-Ouchemoukh et al., 2014)
	2	2.724	196	195.0	Gluconic acid	C6H12O7	59, 71, 74.9, 98.9, 108, 129, 159, 177	-
	3	3.902	494	493.1	Unknown	-	120.5, 194, 209, 243	-
	4	4.209	300	299.0	p-Hydroxybenzoylhexose	C13H16O8	93, 137, 179, 239.02, 239.05	-
	5	4.352	422	421.0	Unknown	-	135, 149, 161, 179, 251, 281, 341	-
	6	4.671	498	497.1	O-dicaffeoyl shikimic acid	C22H25O13	135, 161, 179, 335	Farag et al., 2014
	7	5.224	336	335.2	O-caffeoyl shikimic acid	C16H18O8	131.1, 133, 135, 137, 155, 161, 161.03, 179, 335	Farag et al., 2014
	8	5.294	790	789.2	Unknown	-	389.1, 419.1, 581.1, 743.2	-
	9	5.931	610	609.1	Rutin	C27H30O16	255, 299.9, 301	Abu-Reidah et al., 2013; Farag et al., 2014
	10	6.090	464	463.0	Isoquercetrin	C21H20O12	150.9, 178.9, 255, 270.9, 299.9, 301	Farag et al., 2014
	11	6.213	506	505.2	Isoquercetrin acetate	C23H21O13	150.9, 255, 270.9, 299.9, 301.0, 301.03, 343.2, 505.05, 505.1	Farag et al., 2014
	12	6.306	624	623.2	Isorhamnetin rhamnosyl hexoside	C28H31O16	240.9, 299, 269, 314, 315.02, 315.06, 577, 541, 543, 623.11, 623.19	Farag et al., 2014
	13	6.440	608	607	Unknown	-	284.09, 299.02	-
	14	6.640	510	509.2	Unknown	-	161, 167.1, 221	-
	15	7.035	740	739.3	Unknown	-	119.9, 128, 200, 249, 321, 377.1, 393.1, 409.7, 411.1, 577.2, 649.3, 693.3, 739.2	-
	16	7.843	330	329.2	Ttihydroxy octadecenoic acid	C18H33O5	99.1, 193, 171.1, 211.1, 229.1, 209.1, 293.2, 311.2	Farag et al., 2014
	17	8.095	300	299.0	Chrysoeriol	C16H11O6	255, 256, 284	Farag et al., 2014
24 h	1	1.636	895	894.1	Unknown	-	86.9, 131, 175, 351, 365, 527, 717.6, 805.6, 893.6, 893.8	-
	2	1.875	196	195.0	Gluconic acid	C6H12O7	59, 72.9, 74.9, 86.9, 58.9, 96.9, 129, 159, 177	-
	3	2.167	133	132.9	Malic acid	C4H6O5	71, 114.58	-
	4	2.484	344	343	Unknown	-	161, 163.02, 164, 163.03, 180, 181, 223	-
	5	2.924	584	583.2	Afzelin O-gallate	C28H24O14	135, 164, 179, 251, 281, 341, 403.1, 443.1, 583	(Moharram et al., 2006)
	6	3.453	316	315.0	Protocatechuic acid hexoside	C13H16O9	109, 135, 152.9, 153	Abu-Reidah et al., 2015
	7	3.593	500	499.1	Unknown	-	89, 179, 319.1, 341.07, 341.14, 453.2, 498.8	-
	8	3.949	396	395.0	Unknown	-	96.9, 138.9, 228.9, 240.9	-
	9	4.169	566	565.1	Eriodictyol xyloyldeoxy hexose	C25H26O15	160, 162, 177, 195, 357, 519.1	Abu-Reidah et al., 2015
	10	4.373	300	299.0	p-Hydroxybenzolhexose	-	59, 71, 93, 92, 101, 135.9, 151, 137, 179.01, 179.04, 209, 239.03, 239.05	-
	11	4.481	448	447.2	Luteolin hexoside	C21H19O11	89, 120, 145, 163.05, 163.07, 197.05, 197.04, 285, 447.05, 447.09, 447.15	Farag et al., 2014
	12	4.762	498	497.2	O-dicaffeoyl shikimic acid	C22H25O13	93, 135, 161, 179.01, 179.02, 335.04, 335.01, 497.2, 497.09, 497.12	Farag et al., 2014
	13	5.063	384	383.0	Unknown	-	74.9, 119, 145, 163, 221, 205, 325	-
24 h	14	5.240	450	449.0	Dihydrokaempferol hexoside	C21H22O11	125, 243, 148.9, 259, 269, 287	Abu-Reidah et al., 2015
	15	5.641	476	475.2	Unknown	-	59, 138, 153, 225, 313, 210, 269.1, 475.1, 429.2, 387.1, 475.03	-
	16	5.871	544	543.0	Isoquercetrin sulfate	C21H19O15	150.9, 178.9, 300, 301, 463	Farag et al., 2014
	17	6.151	742	741.2	Unknown	-	179.02, 179.03, 161, 263, 221, 305, 417.1, 433.1, 533.2, 695.3, 741.1, 323	-
	18	6.191	464	463.2	Isoquercetrin	C21H20O12	150.9, 295, 270.9, 299.9, 301, 463.05, 463.13	Farag et al., 2014
	19	6.251	506	505.2	Isoquercetrin acetate	C23H21O13	150.9, 255.03, 255.00, 270.9, 299.9, 301, 343.2, 343.01, 463.04, 503.5, 505.1	Farag et al., 2014
	20	6.458	608	607.1	Chrysoeriol rhamnosyl hexoside	C28H31O15	151, 298, 284, 299, 357.1, 443.1, 607	Farag et al., 2014
	21	6.549	450	449.1	Unknown	-	121, 137, 149, 164.9, 195, 223, 225, 208, 210, 283, 327, 401.1	-
	22	6.675	510	509.2	Unkown	-	89, 179, 167.1, 185.1, 221	-
	23	8.076	326	325.2	p-Coumaroyl hexose	C15H17O8	83, 163.1, 209.1, 307.2, 289	(Amessis-Ouchemoukh et al., 2014)
	24	8.128	542	541.1	Chrysoeriol hexosyl sulfate	C22H21O14S	96.9, 240.9, 241, 241.03, 284.04, 284, 299.02, 299.04, 541.06, 541.02, 541.10	Farag et al., 2014
	25	8.150	300	299.0	Chrysoeriol	C16H11O6	254.9, 256, 284	Farag et al., 2014
	26	8.255	474	473.4	Unknown	-	89, 119, 275.2, 293.2, 311.2, 455.2, 473.2	-
	27	8.958	294	293.1	Unknown	-	131.04, 131.02, 192.09, 205.09, 221.1, 220.1, 236.08, 236.1	-

Table 3. Phytochemical compounds detected after 2, 5 and 24 h of extracted Ajwa date fruit with methanol–water (80%) using LC/ESI-QTOF-MS/MS

Extraction Time	Peak no.	Retention time (min)	Molecular weight	[M-H] m/z	Identified compounds	Molecular formula	Major fragments (MS/MS)	References
2 h	1	1.722	342	341.1	Caffeic acid hexoside	C15H18O9	89.02, 101.02, 179.05, 207.05, 221.06, 251.07, 263.07, 323.09.	-
	2	2.828	500	499.2	Unknown	-	89.02, 161.04, 179.05, 191.1, 341.1, 415.16, 499.16	-
	3	2.953	682	681.2	Unknown	-	101.02, 143.03, 161.04, 179.05, 221.06, 263.07, 323.09, 341.1, 473.16, 501.13, 635.2, 681.2	-
	4	4.378	336	335.1	O-caffeoyl shikimic acid	C16H18O8	132.02, 133.02, 135.04, 155.03, 161.02, 179.03.	Farag et al., 2014
	5	4.422	544	543.2	Isoquercetrin sulfate	C21H19O15	135.04, 179.03, 241, 300.02, 301.03, 335.17, 335.07, 463.09, 497.13, 543.04, 543.16.	Farag et al., 2014
	6	4.462	498	497.1	O-dicaffeoyl shikimic acid	C22H25O13	135.04, 161.02, 179.03, 341.08, 335.07, 361.1.	Farag et al., 2014
	7	4.579	211	210.0	Unknown	-	59.01, 71.01, 87.01, 89.02, 101.02, 113.02, 119.03, 137.02, 165.01, 179.05, 180.04, 195.02.	-
	8	4.751	772	771.3	Quercetin rhamnosyl dihexoside	C33H39O21	299.01, 379.16, 609.15, 608.14, 691.13, 771.21	Farag et al., 2014
	9	4.873	610	609.2	Quercetin7-O-hexoside-3-O-rhamnoside	-	179.05, 284.03, 285.04, 283.02, 327.05, 447.09, 446.09, 489.1, 609.27, 609.15	-
	10	5.065	386	385.2	Sinapic acid hexoside	C17H21O10	59.01, 125.09, 149.02, 205.05, 187.09, 223.06, 265.07, 325.09, 385.04, 385.12, 385.15	Farag et al., 2014
	11	5.710	528	527.0	Unknown	-	241.0, 285.04	-
	12	5.773	286	285.2	Luteolin	C15H9O6	133.04, 151, 133.02, 223.13, 285.13, 285.04, 285.16	(Kim et al., 2009)
	13	6.214	464	463.1	Isoquercetrin	C21H19O12	151.0, 255.03, 271.02, 300.02, 301.03	Farag et al., 2014
	14	6.887	478	477.1	Isorhamnetin-3-O-glucoside	C22H21O12	243.02, 257.04, 271.02, 314.04, 315.05	Farag et al., 2014
	15	8.540	538	537.3	Unknown	-	119.03, 161.04, 179.05, 221.06, 329.23	-
	16	11.007	332	331.3	Unknown	-	185.11, 215.12, 313.24	-
	17	12.906	313	312.2	Unknown	-	183.01, 197.02, 311.17	-
	18	14.054	510	509.3	Unknown	-	152.99, 281.24	-
	19	19.126	326	325.2	Unknown	-	183.01, 184.01	-
	20	20.079	747	746.5	Unknown	-	152.99, 281.24, 255.23, 256.23, 280.23, 279.23, 416.23, 509.29, 510.29	-
	21	20.731	598	597.4	Unknown	-	389.25, 461.29, 535.3, 551.35	-
5 h	1	2.868	460	459.2	Unknown	-	87.05, 128.03, 129.06, 146.04, 200.13, 285.2, 286.14, 330.16, 371.19, 415.18, 459.12	-
	2	3.062	336	335.1	O-caffeoyl shikimic acid	C16H18O8	123.05, 131.08, 137.02, 179.03, 229.08, 239.05, 245.09, 335.07, 335.13	Farag et al., 2014
	3	3.363	775	774.3	Unknown	-	119.04, 163.03, 164.04, 234.07, 294.09, 293.08, 325.09, 326.09, 422.11, 448.15, 447.15, 612.17, 774.15, 774.26	-
	4	3.503	498	497.3	O-dicaffeoyl shikimic acid	C22H25O13	135.04, 137.02, 161.02, 179.03, 195.06, 203.07, 289.07, 335.07, 407.16, 451.22, 497.17, 497.13, 497.25	Farag et al., 2014
	5	4.149	180	179.0	Caffeoyl acid	-	59.01, 71.01, 134.03, 135.04, 136.048	-
	6	5.266	772	771.4	Quercetin rhamnosyl dihexoside	C33H39O21	217.1, 231.05, 249.05, 301.03, 321.07, 411.1, 463.09, 563.23, 563.18, 609.15, 609.19, 691.12, 771.27, 771.22, 725.19, 771.3	Farag et al., 2014
	7	5.558	194	193.0	Ferulic acid	C10H10O4	137.02, 149.06, 133.02, 134.03, 178.02	Yasir et al., 2016
	8	6.081	448	447.1	Leteolin hexoside	C21H19O11	256.03, 285.04, 284.03, 327.05	Farag et al., 2014
	9	6.283	542	541.1	Chrysoeriol hexosyl sulfate	C22H21O14S	241.0, 299.05, 284.03, 299.76	Farag et al., 2014
	10	6.673	478	477.1	Isorhamnetin-3-O-glucoside	C22H21O12	151.0, 242.02, 257.04, 243.02, 271.02, 314.04, 315.05, 357.06	Farag et al., 2014
	11	6.722	608	607.1	Chrysoeriol rhamnosyl hexoside	C28H31O15	298.04, 284.03, 297.03, 299.05	Farag et al., 2014
	12	7.250	806	805.3	Unknown	-	195.06, 225.07, 417.15, 595.22, 643.24	-
	13	8.033	286	285.2	Luteolin	C15H9O6	132.02, 133.02, 151.0, 175.03, 285.04	(Kim et al., 2009)
	14	9.676	464	463.3	Isoquercetrin	C21H19O12	221.18, 239.20, 283.19, 301.2, 343.21, 445.24, 463.09	Farag et al., 2014
	15	9.746	474	473.3	Unknown	-	293.21, 311.22	-
	16	10.601	368	367.2	Unknown	-	157.03, 187.04, 205.05, 277.07, 331.25	-
	17	12.531	313	312.2	Unknown	-	183.01, 197.02, 311.17	-
	18	17.343	326	325.2	Unknown	-	183.01, 279.23	-
	19	19.105	472	471.3	Unknown	-	393.31, 409.35	-
	20	21.391	312	311.2	Unknown	-	183.01	-
	21	23.125	748	747.6	Unknown	-	255.23, 281.25	-
24 h	1	1.731	388	387.1	Caffeic acid hexoside conjugate	-	89.02, 119.03, 179.05, 221.06, 207.04, 251.07, 341.1	-
	2	1.898	478	477.2	Isorhamnetin-3-O-glucoside	C22H21O12	71.01, 134.04, 120.99, 153.01, 195.02, 255.05, 315.07, 417.1, 477.12, 477.17	Farag et al., 2014
24 h	3	2.059	170	169.0	Gallic acid	C7H6O5	97.03, 125.02, 169.01	-
	4	2.493	448	447.2	Luteolin hexoside	C21H19O11	71.01, 120.99, 161.04, 239.12, 225.05, 285.07, 343.11, 401.14, 401.18, 447.15, 447.10, 447.19	Farag et al., 2014
	5	3.077	300	299.1	Unknown	-	136.01, 137.02, 179.03, 239.05, 240.05	-
	6	4.201	498	497.1	O-dicaffeoyl shikimic acid	C22H25O13	135.04, 179.03, 341.08, 335.07	Farag et al., 2014
	7	5.256	610	609.2	Quercetin 3 hexose	-	195.07, 241.0, 301.03, 285.04, 300.02, 327.04, 443.14, 447.09, 563.27, 609.15	-
	8	5.284	544	543.0	Isoquercetrin sulfate	C21H19O15	151.0, 241.0, 301.03, 300.02, 463.09	Farag et al., 2014
	9	5.843	300	299.1	Chrysoeriol	C16H11O6	151.0, 256.03, 284.03	Farag et al., 2014
	10	5.914	514	513.0	Unknown	-	151.0, 178.99, 273.04, 301.03, 433.07	-
	11	6.308	543	542.1	Unknown	-	242.0, 241.0, 285.03, 284.03, 299.05, 300.06	-
	12	6.642	578	577.2	Apigenin 7-O-neohesperidoside	C27H30O14	268.18, 268.03, 269.04, 397.22, 577.07, 577.02	Matsuda, 1966
	13	7.686	226	225.1	Unknown	-	59.01, 71.01, 89.02, 113.02, 179.05	-
	14	7.898	302	301.2	Quercetin	C15H10O7	121.02, 151.0, 178.99, 255.23, 280.98, 301.03	(Shabana et al., 2011)
	15	8.010	286	285.0	Luteolin	C15H10O6	105.02, 133.02, 151.0, 201.01	(Kim et al., 2009)
	16	8.463	226	225.1	Unknown	-	89.02, 71.01, 113.02, 119.03, 207.13	-
	17	9.036	330	329.2	Trihydroxy-octadecenoic acid	C18H33O5	139.11, 183.13, 171.10, 211.13, 229.14, 209.11, 311.22	Farag et al., 2014
	18	9.434	542	541.2	Chrysoeriol hexosyl sulfate	C22H21O14S	96.95, 151.0, 241.0, 284.03, 284.04, 299.05, 377.01, 503.16, 541.07, 541.13	Farag et al., 2014
	19	9.568	368	367.1	Unknown	-	115.02, 157.03, 187.04, 205.05, 277.07, 329.23	-
	20	9.637	464	463.3	Isoquercetrin	C21H19O12	59.03, 138.98, 221.19, 255.19, 301.20, 283.19, 367.1, 463.0, 463.09	Farag et al., 2014
	21	10.604	368	367.1	Unknown	-	115.02, 157.03, 187.04, 277.07, 205.05, 331.24	-
	22	12.196	299	298.1	Unknown	-	119.04, 183.01, 184.01, 197.02, 297.15	-
	23	12.997	313	312.2	Unknown	-	119.05, 183.01, 184.01, 197.02, 311.17	-
	24	13.244	326	325.2	Unknown	-	183.01	-
	25	13.878	341	340.2	Unknown	-	183.01, 184.01	-
	26	15.101	488	487.3	Unknown	-	293.24, 279.23, 321.24	-
	27	18.567	283	282.2	Unknown	-	281.24	-
	28	18.954	341	340.2	Unknown	-	184.01, 183.01, 197.02, 239.07, 339.20	-
	29	19.769	670	669.4	Unknown	-	125.09, 187.09, 255.23, 261.13, 431.23, 499.36	-
	30	23.322	341	340.2	Unknown	-	170.0, 197.02, 183.01, 184.01, 339.20	-

Table 4. Phytochemical compounds detected after 2, 5 and 24 h of extracted Ajwa date fruit with acetone–water (70%) using LC/ESI-QTOF-MS/MS

Extraction time	Peak no.	Retention time (min)	Molecular weight	[M-H] m/z	Identified compounds	Molecular formula	Major fragments (MS/MS)	References
2 h	1	2.690	176	175.1	Ascorbic acid	C6H8O6	59.01, 85.06, 113.06, 115.04, 131.07, 157.04	Yasir et al., 2016
	2	3.234	550	549.2	Unknown	-	89.02, 161.04, 179.05, 179.03, 323.09, 341.12, 383.1, 443.12, 503.17	-
	3	4.178	180	179	Caffeic acid	C9H8O4	59.01, 71.01, 89.02, 134.03, 135.04, 136.04	-
	4	5.135	498	497.2	O-dicaffeoyl shikimic acid	C22H25O13	59.01, 101.02, 131.03, 149.04, 161.04, 191.05, 289.16, 319.17, 317.11, 335.16, 417.15, 451.22, 497.17, 497.1	Farag et al., 2014
	5	5.237	772	771.2	Quercetin rhamnosyl dihexoside	C33H39O21	143.04, 171.07, 200.05, 290.09, 321.07, 411.11, 425.13, 581.22, 563.18, 609.15, 609.18, 691.13, 770.8, 771.37, 771.32, 771.27	Farag et al., 2014
	6	5.897	610	609.1	Rutin	C27H30O16	271.02, 300.02, 301.03	Abu-Reidah et al., 2015
	7	6.697	608	607.2	Chrysoeriol rhamnosyl hexoside	C28H31O15	284.03, 299.05	Farag et al., 2014
	8	7.417	328	327.2	Trihydroxy-9,14-octadecadienoic acid	C18H31O5	89.02, 171.10, 242.98, 229.14, 327.11, 327.18	Farag et al., 2014
	9	7.818	714	713.5	Unknown	-	677.50	-
	10	7.996	464	463	Isoquercetrin	C21H19O12	71.01, 152.99, 193.01, 265.18, 283.19, 300.03, 301.20, 373.12, 463.21	Farag et al., 2014
	11	8.320	774	773.4	Unknown	-	611.33	-
	12	8.973	810	809.3	Unknown	-	150.03, 151.03, 165.05, 195.06, 179.07, 387.14, 417.15, 535.20, 565.21, 613.23, 713.27, 761.29, 791.30, 809.41	-
	13	9.356	329	328.2	Unknown	-	169.12, 183.13, 225.15, 239.16, 291.19, 309.20, 327.22	-
	14	9.726	226	225	Unknown	-	71.01, 89.02, 113.02, 179.05	-
	15	12.493	313	312.2	Unknown	-	119.04, 170, 183.01, 197.02, 311.17	-
	16	13.184	1046	1045.5	Unknown	-	721.42, 835.46, 1000.54, 999.53	-
	17	13.352	296	295.2	Hydroxy octadecadienoic acid	C18H31O3	183.13, 277.21	Farag et al., 2014
	18	13.745	328	327.2	Unknown	-	119.05, 185.02, 183.01, 184.01, 326.19	-
	19	14.576	341	340.2	Unknown	-	183.01, 185.02, 184.01	-
	20	17.266	327	326.2	Unknown	-	119.05, 197.02, 183.01, 184.01, 279.23, 325.18	-
	21	17.639	226	225.1	Unknown	-	89.02, 71.01, 113.02, 119.03, 179.05	-
	22	21.585	313	312.2	Unknown	-	119.04, 197.02, 183.01, 184.01, 311.17, 311.29	-
2 h	23	23.776	795	794.5	Unknown	-	365.26, 364.26, 363.25, 747.63, 729.58, 793.52, 793.63	-
	24	25.758	963	961.7	Unknown	-	89.02, 161.04, 235.08, 255.23, 277.21, 287.07, 379.12, 397.13, 415.14, 565.3, 635.37, 657.35, 675.37, 833.56, 914.61, 913.54, 913.60, 959.61,	-
5 h	1	1.083	377	376.1	Unknown	-	119.03, 142.05, 160.06, 232.08, 250.09, 322.11	-
	2	1.950	237	236.1	Unknown	-	72, 103.05, 145.05, 147.04, 164.07	-
	3	3.777	578	577.2	O-dicaffeoyl shikimic acid acetate	C24H27O14	96.95, 135.04, 179.03, 179.05, 272.08, 329.06, 371.11, 415.15, 497.13, 577.09, 577.19, 577.16	Farag et al., 2014
	4	4.253	336	335.1	O-caffeoyl shikimic acid	C16H18O8	133.02, 135.04, 137.02, 161.02, 179.03, 155.03, 289.07	Farag et al., 2014
	5	4.333	498	497.1	O-dicaffeoyl shikimic acid	C22H25O13	135.04, 179.03, 161.02, 335.07, 361.11, 341.08	Farag et al., 2014
	6	4.603	772	771.3	Quercetin rhamnosyl dihexoside	C33H39O21	195.06, 241.0, 299.02, 379.16, 463.09, 563.18, 609.15, 691.12, 771.2	Farag et al., 2014
	7	4.814	691	690.1	Unknown	-	241.0, 242.0, 285.04, 286.04, 284.0, 448.1, 447.09	-
	8	5.973	464	463.1	Isoquercetrin	C21H19O12	151.0, 255.03, 271.02, 300.03, 301.03	Farag et al., 2014
	9	5.977	594	593.2	Luteolin rhamnosyl hexoside	C27H29O15	179.03, 285.04, 284.03, 384.98, 463.09, 593.15	Farag et al., 2014
	10	5.181	542	541.2	Chrysoeriol hexosyl sulfate	C22H21O14S	96.95, 179.03, 299.05, 298.04, 341.06, 371.07, 407.16, 461.11, 541.21, 541.06, 541.12, 541.25	Farag et al., 2014
	11	6.093	448	447.1	Luteolin hexoside	C21H19O11	285.04, 284.03, 327.05	Farag et al., 2014
	12	6.064	448	447.1	Unknown	-	284.03, 285.04	-
	13	6.246	478	477.2	Isorhamnoetin-3-O-glucoside	C22H21O12	101.02, 135.98, 271.02, 299.01, 300.02, 315.05, 314.04, 329.03, 431.18, 462.08, 477.1, 477.13, 459.2	Farag et al., 2014
	14	6.510	854	853.2	Unknown	-	195.06, 225.07, 389.16, 567.22, 615.25, 645.26, 807.32	-
	15	6.830	462	461.1	Unknown	-	255.02, 283.02, 299.05, 446.08	-
	16	7.168	610	609.2	Unknown	-	166.02, 181.04, 402.13, 417.15, 563.21	-
	17	7.912	610	609.3	Unknown	-	429.25, 447.26	-
	18	9.142	376	375.2	Unknown	-	79.95, 171.1, 211.13, 229.14, 329.23, 357.17, 295.22	-
	19	9.470	330	329.2	Trihydroxy-octadecenoic acid	C18H33O5	99.08, 129.09, 169.12, 201.11, 199.13, 211.13, 229.14, 275.20, 293.21, 311.22, 329.23	Farag et al., 2014
5 h	20	10.424	328	327.2	Trihydroxy-9,14-octadecadienoic acid	C18H31O5	129.09, 171.10, 199.13, 197.11, 242.98, 327.16, 309.2, 309.22, 326.99, 227.12	Farag et al., 2014
	21	11.485	225	224.8	Unknown	-	89.02, 71.01, 113.02, 222.07, 224.09, 59.01	-
	22	13.220	327	326.2	Unknown	-	119.04, 170.0, 183.01, 197.02, 325.18	-
	23	13.800	340	339.2	Unknown	-	170.00, 183.01, 197.02, 293.21	-
	24	18.376	257	256.2	Unknown	-	255.23	-
	25	18.607	328	327.2	Unknown	-	185.00, 282.25, 281.24	-
	26	21.591	313	312.2	Unknown	-	119.05, 197.02, 18.3.01, 184.01, 239.07, 311.17	-
	27	23.281	341	340.2	Unknown	-	184.01, 183.01, 197.02, 339.32, 339.20	-
	28	26.114	787	786.5	Unknown	-	119.03, 179.05, 560.51, 578.52, 668.58, 740.58	-
	29	5.977	594	593.2	Kaempferol-3-O-glucoside-7-O-rhamnoside	-	179.03, 585.04, 284.03, 384.98, 463.09	-
24 h	1	1.223	166	165.0	Unknown	-	75.00, 147.02	-
	2	1.777	354	353.1	Chlorogenic acid	C16H18O9	137.02, 155.03, 173.04, 172.06	Reis et al., 2012
	3	2.656	176	175.0	Ascorbic acid	C6H8O6	85.06, 115.04, 113.06, 157.04	Yasir et al., 2016
	4	3.409	422	421.0	Unknown	-	135.04, 161.02, 179.03, 251.05, 281.06, 341.08	-
	5	3.791	578	577.2	O-dicaffeoyl shikimic acid acetate	C24H27O14	87.00, 135.04, 179.03, 221.04, 241.00, 321.07, 392.02, 497.13, 577.09, 577.15, 577.18, 577.13	Farag et al., 2014
	6	3.911	690	689.3	Unknown	-	152.04, 165.05, 151.03, 241.0, 285.04, 391.12, 392.12, 447.09, 527.18, 599.20, 689.11, 689.24, 689.29	-
	7	4.147	364	363.2	Unknown	-	183.10, 201.11	-
	8	4.818	802	801.2	Unknown	-	367.05, 368.05, 510.09, 639.14, 638.13, 755.21	-
	9	4.903	484	483.2	Unknown	-	89.02, 119.03, 179.05, 221.06, 483.16	-
	10	5.303	498	497.2	O-dicaffeoyl shikimic acid	C22H25O13	89.02, 149.04, 191.04, 273.11, 335.17, 317.01, 407.20, 453.18, 497.17, 497.08	Farag et al., 2014
	11	5.358	392	391.1	Unknown	-	59.01, 201.12, 271.08, 373.18, 391.12	-
	12	5.425	544	543.0	Isoquercetrin sulfate		151.00, 241.00, 301.03, 463.09	Farag et al., 2014
	13	5.791	668	667.1	Unknown	-	241.00, 249.03, 284.03, 299.05, 320.96, 389.16, 425.05, 505.14, 623.24, 667.15, 667.19	-
24 h	14	6.439	572	571.1	Unknown	-	241.00, 329.06, 314.04	-
	15	6.797	608	607.2	Chrysoeriol rhamnosyl hexoside		283.02, 284.03, 299.05, 298.04, 443.10	Farag et al., 2014
	16	7.301	328	327.2	Trihydroxy-9,14-octadecadienoic acid	C18H31O5	127.03, 167.05, 171.09, 183.14, 225.14, 242.98, 265.18, 283.19, 283.11, 309.17, 327.05, 327.11, 327.14	Farag et al., 2014
	17	7.490	342	341.2	Unknown	-	101.02, 209.11, 221.06, 237.05, 236.99, 216.98, 267.06, 282.09, 341.10, 326.08	-
	18	7.519	542	541.3	Chrysoeriol hexosyl sulfate	C22H21O14S	96.96, 179.03, 241.0, 284.03, 299.05, 333.20, 343.04, 495.26, 541.07	Farag et al., 2014
	19	8.009	226	225	Unknown	-	59.01, 71.01, 89.0, 85.03, 101.02, 113.02, 119.03, 143.03, 161.04, 179.05, 180.05, 225.11	-
	20	8.282	614	613.3	Unknown	-	151.03, 165.01, 150.03, 165.05, 195.06, 225.07, 341.1, 357.13, 387.14, 417.15, 520.17, 535.20, 565.21, 613.23	-
	21	8.546	294	293.1	unknown	-	96.56, 79.95, 213.49, 293.10	-
	22	8.887	330	329.2	Trihydroxy-octadecenoic acid	C18H33O5	145.08, 171.10, 211.13, 229.14, 311.22, 329.19	Farag et al., 2014
	23	9.010	300	299.1	Chrysoeriol	C16H11O6	134.03, 255.03, 256.03, 284.03, 283.02, 284.05	Farag et al., 2014
	24	9.690	448	447.3	Luteolin hexoside	C21H19O11	78.95, 119.03, 203.00, 262.99, 285.12, 285.20, 302.98, 322.99, 346.98, 445.79, 447.0, 447.21	Farag et al., 2014
	25	9.753	1149	1148	Unknown	-	869.47, 1147.57	-
	26	10.600	368	367.1	Unknown	-	157.03, 187.04, 185.11, 277.07, 331.24	-
	27	11.460	226	225.1	Unknown	-	59.01, 71.01, 89.02, 113.02, 119.03, 149.04, 161.04, 179.05, 194.08, 224.09, 224.80	-
	28	13.117	328	327.2	Pinenol-O-glucoside	C18H31O5	119.04, 171.0, 183.01, 184.01, 185.0, 242.98, 326.18	Farag et al., 2014
	29	13.826	341	340.2	Unknown	-	170.0, 183.01, 184.01, 294.21	-
	30	14.473	488	487.3	Unknown	-	113.02, 161.04, 275.23, 279.23, 293.24, 321.24, 351.25, 441.28	-
	31	15.507	454	453.3	Unknown	-	134.95, 255.23, 371.24, 389.25, 407.26, 453.01	-
	32	17.254	327	326.2	Unknown	-	183.01, 184.01, 279.73, 325.18	-
	33	17.525	340	339.2	Unknown	-	183.01, 184.02, 197.02	-
	34	18.522	328	327.2	Unknown	-	185.0, 282.25, 281.25	-
24 h	35	18.906	472	471.3	Unknown	-	61.99, 393.31, 409.35	-
	36	21.626	313	312.0	Unknown	-	119.04, 197.02, 183.01, 184.01, 311.29, 311.17	-
	37	22.693	751	750.5	Unknown	-	119.03, 179.05, 255.23, 283.26, 512.31, 532.48, 704.55, 714.55, 712.54	-
	38	23.289	341	340.2	Unknown	-	184.01, 183.01, 197.02, 339.32, 339.20	-

Conclusion

The study found date of Ajwa cultivar rich in phytochemicals. Polyphenols could be major phytochemicals identified in this studied date. Interestingly, 21 compounds were identified using UHPLC-ESI-QTOF-MS/MS for the first time. Further works are needed to quantify these compounds. Extraction of phytochemicals using aqueous-based solvents for 24 h exhibited high yield.

Additional information

Funding

This study was fully funded by the Ministry of Higher Education Malaysia [USIM/NRGS_ P10/FPSK/ 8410/52113]. OAN was supported by a postgraduate studentship from the Ministry of Higher Education of Iraq.