Accurate Determination of 24 Water-Soluble Synthetic Colorants in Premade Cocktail Using Ultra-Performance Liquid Chromatography with Diode Array Detection

Abstract

A rapid, traceable, and highly sensitive method was developed for the simultaneous separation and quantification of 24 water-soluble synthetic colorants in premade cocktails, utilizing ultra-performance liquid chromatography coupled with diode array detection (UPLC-DAD). The purity of each colorant was individually confirmed through multi-wavelength analysis. Chromatographic conditions, including mobile phase composition and gradient elution, were meticulously optimized, achieving the separation of the 24 colorants on a BEH C18 column using a linear gradient elution within 16 min. The mobile phase consisted of an ammonium acetate solution (100 mmol/L, pH 6.25) and a mixed organic solvent of methanol and acetonitrile (2:8, v/v). The method exhibited excellent linearity across the concentration range of 0.005–10 μg/mL, with limits of detection (LODs) ranging from 0.66 to 27.78 μg/L for all 24 colorants. The method also demonstrated good precision (0.1–4.9%) at various concentration levels and recoveries ranging from 87.8% to 104.5% at spiked concentrations of 0.1, 0.5, and 1.0 μg/mL. A comparison with other published methods for colorant determination in food samples using HPLC-DAD and LC-MS (2014–2024) revealed that the proposed method offers superior performance in terms of the number of analytes detected, lower limits of detection, and reduced analytical time. Finally, the method was successfully applied to the analysis of colorants in premade cocktails from different sources.

1. Introduction

In the contemporary food industry, colorants are extensively used to enhance the aesthetic appeal of a wide range of products, including beverages and premade cocktails. These additives impart vibrant colors that significantly improve the visual attractiveness of food, which, in turn, can positively influence consumer preferences and boost sales [1]. Colorants utilized in the food industry are broadly categorized into synthetic and natural dyes. Compared to natural colorants, synthetic colorants are more cost-effective, more stable, and more accessible. Therefore, the application of synthetic food colorants has become increasingly prevalent in the food industry [2]. However, some food colorants posed a potential risk to human health, especially if they were excessively consumed. For this reason, safety data, such as the acceptable daily intake, based on toxicological studies [3,4] on experimental animals and human clinical studies, have been repeatedly determined and evaluated by the FAO and WHO [5,6].

To protect public health, many countries have established strict regulations regarding the permissible types, application ranges, and maximum allowable concentrations of food colorants [7,8,9]. Nonetheless, some food producers exceed these limits or introduce illegal colorants, which can pose significant health risks [10,11]. At the same time, all food ingredients and content including food colorants are required to be listed on the food labels, and these ingredients need be the same as those on the food labels [12]. Consequently, it is crucial to develop rapid and accurate methods for the simultaneously determination of food colorants in various samples in order to ensure compliance with food safety standards and to meet the needs of this expanding food market, particularly in China.

Various analytical methods for determining food colorants have been proposed, including spectrophotometry [13], Raman spectroscopy [14], capillary electrophoresis (CE) [15], voltammetry [16,17], fluorescence quenching method [18], ion mobility spectrometry [19], high-performance liquid chromatography (HPLC) with ultraviolet/visible (UV/Vis) or diode-array detector (DAD) detection [20,21,22,23,24,25,26,27,28,29,30], and liquid chromatography tandem mass spectrometry (LC–MS/MS) [31,32,33,34,35,36,37,38].

While voltammetry, spectrophotometry, and Raman spectroscopy were simple and rapid, they are less suitable for the simultaneous detection of multiple colorants. Capillary electrophoresis offers high resolution, short analysis time, and low sample and reagent consumption, making it effective for analyzing complex matrices, though it exhibits poorer repeatability compared to HPLC. Ion mobility spectrometry and fluorescence quenching methods are less commonly applied due to inherent limitations of their own, and research on the analysis of food colorants using these methods is in its infancy, requiring further development.

Traditional techniques such as HPLC-DAD and LC–MS/MS remain widely used for the simultaneous analysis of numerous colorants, with recent advancements in liquid chromatography high-resolution mass spectrometry (LC-HR-MS) offering better selectivity, better sensitivity, and the possibility for multi-analyte detection [38,39]. Therefore, LC-HR-MS is particularly valuable for screening colorants in food or detecting new illegal additives, such as Rhodamine 6G, for which standard reference materials are not yet available [32]. In the LC-MS/MS methods, small amounts of organic acids, such as formic acid or trifluoroacetic acid, are typically added to the mobile phase to enhance the ionization of the ESI ion source. However, the pH sensitivity of colorants during ionization can lead to the formation of multi-charged or unstable ions, often necessitating frequent cleaning of the ion source [33,34]. Despite its high initial costs and matrix interference challenges, LC–MS/MS remains indispensable for the multi-analyte analysis of food colorants.

The HPLC-DAD method benefits from the strong absorption peaks of food colorants within the visible wavelength (400–700) nm. By optimizing the gradient elution program and selecting appropriate multi wavelength monitoring mode in the DAD, this method enables effective analysis of numerous colorants [20,21,22,23,24,25,26,27,28,29,30,39]. In recent years, due to the trace level of food dyes and the complexity of food matrices, sample pretreatment has become an inevitable procedure prior to instrumental analysis in order to enhance the sensitivity through pre-concentration. Recent HPLC studies have utilized techniques like liquid–liquid extraction (LLE) [21,23,33,36,40], solid-phase extraction (SPE) [28,35], dispersive solid-phase extraction (d-SPE) [24,25,34,41], and ultrasound-assisted extraction (UAE) [30] for isolating colorants. While these methods provide excellent quantitative results, they typically permit the simultaneous analysis of fewer than ten colorants, limiting their utility for monitoring synthetic dyes in commercially available foods.

Compared to LC-MS/MS, HPLC-DAD is more cost-effective, simpler, and easier to implement in routine food analysis [22,23,24,25,26,27,28,29]. Following the introduction of GB 5009.35-2023 [39], research into the chromatographic analysis of food colorants has become a prominent focus in China. Thus, the development of methods for the simultaneous analysis of various synthetic colorants using HPLC-DAD, with low cost, simplicity, and a short analysis time, remains a promising area of research.

However, the simultaneous determination of a large number of structurally diverse colorants remains a major analytical challenge due to their differing acid–base properties, solubilities, and polarities. Therefore, the present work employed a cost-effective HPLC approach that is suitable for routine use in most analytical laboratories, while achieving an extended linear range and demonstrating improved performance in terms of LOD and LOQ compared to previously reported methods.

The present study aimed to develop a practical and versatile multiclass HPLC-DAD method, alongside optimized sample preparation, for the simultaneous determination of a diverse range of food colorants with varying acidic–basic properties, solubility, and polarities in premade cocktails samples. This study reports a rapid and efficient method for detecting 24 food colorants, including illegal ones, using UPLC-DAD, contributing to advancements in colorant analysis and facilitates colorant monitoring and regulation in the food industry. The analyzed colorants include both permitted and prohibited substances across different countries. Notably, colorants such as Ponceau SX, Ponceau S, Red 2 G, and Rhodamine B, which were once approved for use in food, are currently banned due to their toxicity [6]. The developed method was validated according to the GB2760 and FDA guidelines for chemical methods validation [8,9] and was applied to analyze approximately 100 premade cocktail samples collected from local markets in China.

2. Experimental Section

2.1. Chemicals and Reagents

The details of the 24 food colorants reference materials (RM) are presented in

Table 1. The information of 24 water-soluble synthetic colorants.

Name	Molecular Formula	No. CI	CAS Number	MW	λ (max) a	Detection Wavelength	Purity (g/g) b	Provider/CRM Number c	Solubility d
Tartrazine	C16H9N4Na3O9S2	19140	1934-21-0	534.36	427 nm	420 nm	/	NIM, GBW(E)100001a 0.5 mg/mL ± 1%	260 g/L
Amaranth	C20H11N2Na3O10S3	16185	915-67-3	604.47	521 nm	520 nm	/	NIM, GBW(E)100002a 0.5 mg/mL ± 2%	50 g/L
Ponceau 4R	C20H11N2Na3O10S3	16255	2611-82-7	604.47	508 nm	520 nm	/	NIM, GBW(E)100004a 0.5 mg/mL ± 2%	25 g/L
Sunset yellow	C16H10N2Na2O7S2	15985	2783-94-0	452.37	483 nm	490 nm	/	NIM, GBW(E)100003a 0.5 mg/mL ± 1%	50–100 g/L
Allura red AC	C18H14N2Na2O8S2	16035	25956-17-6	496.42	509 nm	520 nm	/	NIM, GBW(E)100192 0.5 mg/mL ± 2%	53.878 g/L
Brilliant blue	C37H34N2Na2O9S3	42090	3844-45-9	792.85	629 nm	620 nm	/	NIM, GBW(E)100005a 0.5 mg/mL ± 2%	25 g/L
Crystal violet	C25H30ClN3	42555	548-62-9	407.99	590 nm	520 nm	98.1% ± 1%	NIM, GBW06420 98.1% ± 1%	16 g/L
Erythrosine	C20H6I4Na2O5	45430	568-63-8	879.86	526 nm	520 nm	/	NIM, GBW(E)100191 0.2 mg/mL ± 2%	100 g/L
Rhodamine B	C28H31ClN2O3	45170	81-88-9	479.01	550 nm	520 nm	99.0% ± 0.4%	NIM, GBW06146 99.0% ± 0.4%	10 g/L
Orange G	C16H10N2Na2O7S2	16230	1936-15-8	452.37	480 nm	490 nm	98.5% ± 0.5%	Sigma–Aldrich	50 g/L
Ponceau S	C22H12N4Na4O13S4	27195	6226-79-5	760.57	520 nm	520 nm	98.5% ± 0.6%	Sigma–Aldrich	10 g/L
Red 2G	C18H13N3Na2O8S2	18050	3734-67-6	509.42	532 nm	520 nm	98.3% ± 0.8%	Sigma–Aldrich
Azorubine	C20H12N2Na2O7S2	14720	3567-69-9	502.43	518 nm	520 nm	98.8% ± 0.8%	Sigma–Aldrich
Ponceau SX	C18H14N2Na2O7S2	14700	4548-53-2	480.42	504 nm	520 nm	96.6% ± 1.6%	Sigma–Aldrich
Orange II	C16H11N2NaO4S	15510	633-96-5	350.32	485 nm	490 nm	98.6% ± 0.7%	Toronto Research Chemicals	116 g/L
Orange IV	C18H14N3NaO3S	13080	554-73-4	375.38	428 nm	420 nm	98.5% ± 0.6%	Toronto Research Chemicals
Metanil yellow orange MNO	C18H14N3NaO3S	13065	587-98-4	375.38	420 nm	420 nm	98.4% ± 1.6%	Toronto Research Chemicals	25 g/L
Chrysoidine Y	C12H13ClN4	11270	532-82-1	248.71	408 nm	420 nm	99.0% ± 0.8%	Sigma–Aldrich
Fast green FCF	C37H34N2O10S3Na2	42053	2353-45-9	808.86	623 nm	620 nm	96.2% ± 2.0%	Toronto Research Chemicals
Brilliant green	C27H34N2O4S	42040	633-03-4	482.63	626 nm	620 nm	97.2% ± 1.4%	Toronto Research Chemicals	100 g/L
Acid red 52	C27H29N2NaO7S2	45100	3520-42-1	580.65	562 nm	520 nm	96.0% ± 2.0%	Sigma–Aldrich
Naphthol yellow	C10H4N2Na2O8S	10316	846-70-8	358.19	431 nm	420 nm	99.3% ± 0.7%	Dr. Ehrenstorfer
Neutral Red	C15H17ClN4	50040	553-24-2	288.78	461 nm	490 nm	95.8% ± 2.4%	Dr. Ehrenstorfer	50 g/L
Uranine	C20H10Na2O5	45350	518-47-8	376.27	488 nm	490 nm	99.6% ± 0.4%	Dr. Ehrenstorfer	500 g/L

^a The data for the maximum absorption wavelength was from Merck product manual. ^b For the 24 colorant RMs, except the 9 CRMs obtained from NIM, the purity was determined by UPLC-DAD at different wavelengths corresponding to the maximum absorption of colorants (420 nm, 490 nm, 520 nm, 620 nm). The purity values were expressed as the measurement result plus the standard deviation. ^c NIM: National Institute of Metrology for China; GBW or GBW(E): National certified reference materials in China. ^d Solubility refers to the solubility in water at temperature of 20–25°C.

. Nine certified reference materials (CRM) were provided by the National Institute of Metrology, China (NIM), seven RMs were purchased from Sigma–Aldrich company (St. Louis, MO, USA), five from Toronto Research Chemicals Inc. (Toronto, ON, Canada), and three from Dr. Ehrenstorfer company (Augsburg, Germany). All colorant RMs were used as received and with purities exceeding 85%, as indicated in their respective certificates. Additionally, for all colorant RMs, except the nine CRM obtained from NIM, the purity was re-evaluated using the HPLC-DAD method, and the results are displayed in Table 1.

HPLC-grade formic acid was purchased from Acros Organics Company (Fair Lawn, New Jersey, USA). Acetic acid (HPLC grade) was obtained from CNW Technologies GmbH (Duesseldorf, Germany). Ammonium acetate, methanol, and acetonitrile (HPLC grade) were purchased from Merck Company (Darmstadt, Germany). Water was purified through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Ammonia solution (Analytical pure) was obtained from Beijing Chemical Works (Beijing, China).

2.2. Standard Solution Preparation

A stock solution of 100 μg/mL, containing 17 water-soluble synthetic colorants (except for the seven CRM colorants obtained from NIM), was prepared using ultrapure water Recovery studios (18.2 MΩ·cm) from a Milli-Q system (Millipore, USA). Each synthetic colorant was accurately weighed using a balance (0.01 mg) according to the purities listed in Table 1.

Mixed standard solutions, containing 24 water-soluble synthetic colorants at a concentration of 50 μg/mL, were freshly prepared from stock solutions and the solutions from NIM. Through step-by-step dilution, a series of calibration solutions was prepared, with concentrations of 0.01, 0.05, 0.1, 1.0, 5.0, 10.0, and 20.0 μg/mL. Recovery studies were conducted on real matrix samples spiked with known concentrations of the analytes. All solutions were stored at 4 °C in the dark and remained stable for seven days. All of the above solution preparations were carried out under light-protected conditions.

2.3. Instrumentation and Conditions

The analysis was performed using a Dionex Ultimate 3000 liquid chromatography system (Waltham, MA, USA), equipped with high-pressure ternary pumps, a auto-sampler, a thermostatic column compartment, and a diode array detector. Chromeleon 7.0 software was utilized for system operation and data analysis. Separations were achieved on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm × 1.7 μm; Waters, Milford, CT, USA).

The mobile phase system consisted of A (100 mmol/L ammonium acetate aqueous solution, adjusted pH to 6.25 with acetic acid) and B (methanol/acetronitrile, v/v, 2/8), with a gradient elution as follows: (0~12) min 4%~50% B, (12~12.1) min 50%~95% B, (12.1~16.5) min 96% B, and (16.5~17) min 96%~4% B, followed by a return to initial conditions in 0.5 min. The flow rate was set to 0.3 mL/min, with column and auto-sampler temperatures maintained at 35 °C and 10 °C, respectively. The sample injection volume was 5 μL. The column eluents were monitored at 420, 490, 520, and 620 nm, depending on the colorant, and the absorption spectra of the food colorants were recorded between 400 and 700 nm. Peak identification was performed by comparing the retention times and absorption spectra of the samples with those of the food colorants standards.

2.4. Sample Preparation

Approximately 100 premade cocktail samples from various brands were purchased from a local market in China. For premade cocktails sample preparation, 15 mL of each cocktail was transferred to a centrifuge tube and centrifuged for 5 min at 8000 rpm, and the supernatant (10 mL) was transferred to in another centrifuge tube. The samples were then evaporated in a 40 °C water-bath under a nitrogen flow for 10 min to remove the ethanol and degassed by ultrasonication for 10 min to remove the carbon dioxide. After cooling to room temperature, the evaporated volume was replenished with water. The sample solution was adjusted to pH 6.5 using ammonia solution before solid phase extraction (SPE) on an HLB cartridge (500 mg, Waters, Milford, CT, USA).

The SPE cartridges were preconditioned with 5.0 mL methanol followed by 5.0 mL acidified water (contained 0.1% (v/v) formic acid). Samples were loaded and passed through the cartridges at a rate of 1.0 mL/min. The cartridges were then rinsed with 5.0 mL of 15% (v/v) methanol/water solution (the water contained 0.1% formic acid) and were finally eluted with 3.0 mL of methanol, followed by 3.0 mL methanol containing 5% (v/v) ammonia. The eluate was dried at 40 °C under a gentle nitrogen gas flow, and the residue was reconstituted in 1 mL with water. All of the above preparations were carried out under light-protected conditions as soon as possible (Scheme 1).

2.5. Method Validation

Quantitative analysis was performed using the external standard calibration method. Calibration solutions were prepared through appropriate dilutions of standard solutions to achieve concentrations ranging from 0.01 to 50 μg/mL. The limit of detection (LOD) and the limit of quantitation (LOQ) were evaluated based on the minimum concentrations that produced signal-to-noise (S/N) ratios of 3 and 10, respectively.

Repeatability (intra-day precision) was evaluated by analyzing standard solutions at concentrations of 0.5, 5.0, and 10.0 μg/mL, each performed six times in one day, and reproducibility (inter-day precision) was assessed by independent analysis conducted three times by two analysts over five different days. Recovery experiments were performed using blank premade cocktail samples spiked with 24 colorants at concentrations of 0.1, 0.5, and 1.0 μg/mL. Each spiked sample was analyzed six times at each concentration level.

3. Results and Discussion

3.1. HPLC-DAD Method Development

To optimize chromatographic conditions, key factors, such as column type, mobile phase composition (aqueous concentration and pH), and gradient elution program, were systematically investigated. Optimal separation conditions for the HPLC-DAD analysis were established to achieve the best resolution in the shortest analysis time. Four columns were tested, including Acquity UPLC BEH C18 columns (2.1 × 50 mm, 1.7 μm and 2.1 × 100 mm, 1.7 μm) from Waters, the Zorbax SB-C18 column (4.6 × 50 mm, 1.8 μm) from Agilent, and the Shim-pack XR-C18 column (3.0 × 75 mm, 2.2 μm) from Shimadzu. After adjusting the flow rates and gradients for each column, the ACQUITY UPLC BEH C18 column was found to effectively separate 24 synthetic colorants within 16 min, especially for challenging colorants such as Neutral Red, Erythrosine, Uranine, and Azorubine, achieved the best resolution (Supplementary Materials Table S1 and Figure S1).

3.1.1. Mobile Phase

In previous liquid chromatography (LC) analyses utilizing ammonium acetate buffer acetonitrile (ACN) as the mobile phase, acidic colorants were eluted before basic and neutral ones [18,38]. Consistent with these observations, we also observed that acidic dyes, such as Acid Red 33, were eluted earlier in this chromatographic system. Sun, H.W et al. employed a similar BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) identical to the one employed herein to effectively separate 21 dyes over a 15 min period, with the mobile phase comprising 20 mM ammonium acetate and 0.02% acetic acid (pH 5) with CAN [27]. Other studies have also reported successful separation of dyes using ammonium acetate buffer and ACN as the mobile phase [22,25,28,38,39].

To optimize the aqueous phase, the concentration and pH of the ammonium acetate solution were varied. Three concentrations of ammonium acetate solution (50, 100, and 150 mmol/L) were tested, and the results indicated that increasing the concentration did not significantly affect the peak areas of the colorants. However, the retention times and resolutions of several colorants were notably influenced by the concentration of ammonium acetate aqueous solution (Figure 1). The resolutions of Sunset yellow, Uranine, and Neutral Red improved with increasing concentration, whereas the resolutions of Ponceau 4R, Metanil yellow orange MNO, and Rhodamine B decreased. Some colorants, such as Orange G, Red 2G, and Acid Red 52, exhibited an initial increase in resolution followed by a decrease. Consequently, the optimal concentration of ammonium acetate solution for achieving the maximum resolutions of Sunset yellow, Uranine, and Neutral Red, without compromising the resolution of other analytes, was determined to be 100 mmol/L.

The pH of ammonium acetate aqueous solution had a significant effect on the separation of Uranine, Erythrosine, and their adjacent colors. As shown on Figure 2, the retention times of Uranine and Erythrosine increases with decreasing pH values. At a pH of 6.31, Neutral Red and Erythrosine could not be completely separated. When the pH was lowered to 6.07, Neutral Red and Erythrosine were successfully separated, but Uranine and Azorubine exhibited similar retention times. Ultimately, a pH of 6.25 for mobile phase A was selected as optimal as it allowed for the complete separation of Uranine, Azorubine, Neutral Red, and Erythrosine.

The mobile phase composition of methanol/acetonitrile = 2:8 (v/v) was selected based on a comprehensive evaluation of its effects on both retention time and chromatographic resolution. An increase in methanol proportion resulted in prolonged retention times and varying impacts on resolution among different colorants, enhancing the separation of certain analytes while diminishing that of others. To balance analysis efficiency with separation performance, particularly for pigments exhibiting lower resolution or sensitivity to organic phase composition, the ratio of methanol to acetonitrile at 2/8 (v/v) was determined to be optimal and was thus employed in subsequent analyses.

For mobile phase B, common reversed-phase solvents such as methanol, acetonitrile, and a methanol–acetonitrile mixture were compared. A methanol–acetonitrile ratio of 2/8, (v/v) provided the best peak separation.

3.1.2. Gradient Program

A well-designed gradient program is essential for achieving fast and efficient separation and complex mixtures. Given the diverse nature of the colorants and their varying retention behaviors, isocratic elution was insufficient for effective separation. Therefore, gradient elution was employed to enhance separation efficiency. The chromatographic separation of 24 water-soluble synthetic pigments under six different gradient conditions (Gradient 1–6) is shown in Figure 3.

Six typical gradient elution programs were evaluated to optimize the separation conditions. These included various initial and final proportions of mobile phase B and different gradient durations. Specifically, Gradient 1 employed a linear increase from 4% to 50% B over 12 min, followed by a rapid ramp to 96% B and a hold phase and then re-equilibration. Gradient 2 started at 5% B and increased to 55% over 12 min, followed by a step to 90% B and a subsequent hold. Gradient 3 involved a transition from 5% to 47% B over 12 min, a sharp increase to 95% B, and a prolonged elution at high organic content. Gradient 4 and Gradient 5 both featured extended linear gradients to 65% and 70% B, respectively, before increasing to 95% B. Gradient 6 consisted of a multi-stage profile, beginning with a ramp from 5% to 40% B, an isocratic hold, and subsequent steps to 65% and 95% B, before returning to initial conditions. These gradient programs were systematically compared to identify the most suitable conditions for efficient and reproducible separation of the target analytes.

Each synthetic colorant exhibited a unique maximum absorption wavelength. The quantitation wavelengths for the colorants were selected based on their respective absorption maxima, with 420 nm, 490 nm, 520 nm, and 620 nm chosen for monitoring. Under these optimization conditions, baseline separation of 24 colorants was achieved within 16 min. The resolution value ranged from 2.36 to 19.32, well above the theoretical minimum value of 1.5. The peak symmetry ranged from 1.06 to 1.71, fully meeting the requirements for quantitative analysis (see

Table 2. Summary of experience on adding colorants in premade cocktails.

Flavor in Label of Premade Cocktails	Color of Premade Cocktails	Commonly Detected Colorants
Strawberry	Red	Amaranth, Ponceau 4R, Allura red AC
Peach	Pink	Amaranth, Ponceau 4R,
Orange	Orange yellow	Tartrazine, Sunset yellow, Allura red AC
Pineapple	Yellow	Tartrazine, Sunset yellow
Mango	Golden yellow	Amaranth, Tartrazine
Grape	Purple	Amaranth, Brilliant blue
Breezer	Green	Tartrazine, Brilliant blue
Apple	Cyan	Tartrazine, Sunset yellow Brilliant blue
Blueberry	Blue	Brilliant blue

).

The UPLC chromatogram of the mixed synthetic colorant standard solution is shown in Figure 4, with colorants identified by their retention times and maximum absorption wavelengths.

3.2. Method Validation Result

The linearity for each colorant was evaluated by analyzing mixed standard solutions of varying concentrations (0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 8.0, 10.0, 20, and 50 μg/mL). The analyses were performed in triplicate, with quantitation being based on the peak area. As shown in Table 2, all colorants exhibited excellent correlation coefficients (R² > 0.9980) within their respective linear range. The limits of detection (LOD) and quantitation (LOQ) were defined as three and ten times the signal-to-noise ratio, respectively. The LOD ranged from 0.66 μg/L (Brilliant blue) to 27.78 μg/L (Neutral Red). The LOD ranged from 0.66 μg/L (Brilliant blue) to 27.78 μg/L (Neutral Red). Compared to the detection limits previously reported in the literature [22,23,26,39], the sensitivity of this method was improved by a factor of three or more.

Method precision was assessed by evaluating the variation in peak areas both within a single day (intra-day precision) and across multiple days (inter-day precision). Intra-day repeatability was examined by analyzing standard solutions of 0.5 μg/mL, 5 μg/mL, and 10 μg/mL, each in seven replicates, on the same day. Inter-day reproducibility was assessed by independent analysis, performed by two analysts, across five separate days. The precision data, shown in Table 2, reveal that intra-day relative standard deviations (RSD) ranged from 0.11% to 3.33% (n = 7), while the inter-day RSD values ranged from 0.89% to 5.44% (n = 5). These results demonstrate that the method’s precision is robust and suitable for its intended purpose.

Recovery studies were conducted at dye concentrations of 0.1 μg/mL, 0.5 μg/mL, and 1.0 μg/mL (Supplementary Materials Table S2). Recoveries (n = 6) in premade mixtures ranged from 75.01% to 114.45%, with RSD values ranging from 0.19% to 5.95%. The recovery and the precision data of spiked recovery provided by the developed analytical method meet the requirements of GB 2760; Standard’s Title: National Food Safety Standard—Standards for Uses of Food Additives. Publisher: National Health Commission, Beijing, China, 2024and GB 5009.35; Standard’s Title: National Food Safety Standard—Determination of Synthetic Colour in Foods. Publisher: National Health Commission, Beijing, China, 2023. The recovery is typically required to be between 70% and 115%. The colorant detection standards for repeatability should be relative standard deviation (RSD) ≤ 10%. The intra-day precision and inter-day precision for 24 colorants see Table S3.

Notably, recoveries for Alkaline Chrysoidine Y (Peak 21), Bright green (Peak 24), and Crystal violet (Peak23) were lower and were not subjected to statistical analysis. The recoveries of these three pigments are below the requirement of GB 5009.35-2023 (70–115%), but this does not affect the applicability of the method. First, these three pigments are not contained in the list of permitted synthetic colorants specified in GB 2760-2024, which are considered illegal additives. Second, the 24 water-soluble colorants in the paper covered by include commonly synthetic colorants. The precision and LOD of the developed analytical method are superior to the requirements of the GB 5009.35-2023. This observation is likely attributable to the cationic nature of these pigments, which contain positively charged chromophores. The premade cocktails contained organic acids, such as citric acid, which are prone to generating negative charge. Consequently, the positive charged chromophores of these colorants may bind to the negatively charged citric acid molecules, potentially hindering their retention on the extraction column. This behavior is consistent with previous studies reporting the efficient adsorption of Crystal violet by sorghum straw modified with citric acid for wastewater treatment [42].

Over 100 commercially available premade cocktail samples with varying prices were analyzed using the established UPLC-DAD method. In these samples, seven synthetic colorants permitted by the GB 2760 were detected. The concentration ranges for these colorants were as follows: Allura red AC (2.05–10.62 μg/mL), Amaranth (1.82–10.77 μg/mL), Ponceau 4R (0.72–3.88 μg/mL), Tartrazine (0.04–0.87 μg/mL), Sunset yellow (1.74–9.80 μg/mL), Brilliant blue (0.12–3.48 μg/mL), and Erythrosine (0.12–2.34 μg/mL). The testing frequencies for the colorants were as follows: Allura red AC (18), Amaranth (26), Ponceau 4R (16), Tartrazine (46), Sunset yellow (20), Brilliant blue (56), and Erythrosine (10) (see Figure 5).

Among all samples, approximately 30% contained three colorants simultaneously, about 40% contained two colorants simultaneously, and roughly 30% contained one colorant. No instances of prohibited synthetic colorants or instances exceeding the allowable limits of single colorants were observed. Finally, through data analysis and comparison with the compositions listed on the product labels, some samples were found to have discrepancies between the colorant compositions listed on the product labels and those actually detected, indicating non-compliance with GB 7718 requirements.

Moreover, extensive testing and market research revealed that customers, particularly younger individuals, often judge the quality of premade cocktails based on visual impressions of color rather than actual fruit flavors. The flavor profiles of these cocktail are influenced more by added colorants than by the authentic fruit taste. Notably, the incorporation of colorants followed a consistent pattern, as demonstrated in Table 2. This highlights the necessity for enhancing the regulation of cocktail samples to ensure consumer safety.

3.3. Comparison with Published Results

For comparison with published methods (Table S4 in the Supplementary Materials), Table S4 lists the papers published on colorant detection methods (2014–2024) alongside the method presented in this paper. In comparison with published methods, the limits of detection, number of colorants, precision, and recovery of the developed method exceeded those reported in the references [24,25,28,35,36,43]. Thus, the present method represents a significant advancement in the measurement of target analytes in cocktail samples, particularly for Tartrazine, Ponceau 4R, Red 2G, Azorubine, and Brilliant blue.

4. Conclusions

This study proposes a novel method for the simultaneous determination of 24 water-soluble synthetic colorants in premade cocktails, employing ultra-performance liquid chromatography coupled with diode array detection (UPLC-DAD) following a solid-phase extraction (SPE) pretreatment. The purity of each colorant was verified through multi-wavelength analysis to ensure result traceability and analytical accuracy. The method exhibited excellent linearity and low limits of detection (LODs). Compared to methods published between 2014 and 2024 (Table S4) [22,23,24,25,33,34,35,36,37,41,43], the developed approach demonstrates superior performance regarding the number of analytes, detection sensitivity, and analytical throughput.

Utilizing this method, over 100 premade cocktail samples were analyzed. Extensive sample testing combined with market research revealed that consumers primarily assess product quality based on the visual perception of color. Notably, the flavor profiles of premade cocktails were found to be influenced more by the colorants added than by the taste of natural fruits. A systematic pattern in the usage of synthetic colorants across different products was also identified.