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Background: SARS-CoV-2 variant tracking is key to the genomic surveillance of the COVID-19 pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification. Case presentation: A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike amino acids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation. Conclusions: To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the probe target area of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.

Background: SARS-CoV-2 variant tracking is key to the genomic surveillance of the pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification.Case presentation: A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike aminoacids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation. Conclusions: To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the targets of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.

A reliable and rapid capillary zone electrophoresis method for the determination of the major of cations in milk samples was developed. Sample preparation consisted of dilution, acidification to pH 4.0 with 1 mol L −1 acetic acid and filtration. The complete separation of K + , Ca 2+ , Na + and Mg 2+ could be achieved in 4 min with a simple electrolyte composed of 10 mmol L −1 imidazole and 1 mol L −1 acetic acid (pH 3.6). The running voltage was 25 kV at 25 °C. Indirect UV detection was achieved at 185 nm. Detection limits ranged from 0.06 to 0.57 mg L −1 and quantification limits ranged from 0.16 to 0.62 mg L −1 . Precision data showed relative standard deviations (RSD%) lower than 4.1% for relative migration time and 2.4% for milk concentrations, respectively. Recoveries of cations in samples analysed ranged from 97.7% to 101.1%. Thirty samples of milk were analysed, obtaining mean values of 1.46, 1.19, 0.505 and 0.126 g L −1 for K + , Ca 2+ , Na + and Mg 2+ , respectively.

An accurate, simple and rapid capillary zone electrophoresis (CZE) method with direct UV detection has been set up for the determination of main organic acids in grape juice and wine. The determination of tartaric, malic, and citric acids in grape juices and tartaric, malic, succinic, acetic, lactic and citric acids in wines can be achieved in less than 3 min with only a simple dilution and filtration treatment of the sample. Validation parameters of the method as detection and quantification limits, linearity, precision (intraday and interday analysis) and recovery were also studied in grape juice, white wine, rose wine and red wine, separately. The proposed method decreases the analysis times of the previous reported CZE methods and allows the rapid control of the grape maturity, the winemaking processes and the detection of wine alterations and/or illnesses.

A capillary zone electrophoresis method for the determination of inorganic anions and formic acid in honey samples was developed for the first time. The complete separation of chloride, nitrate, sulfate, phosphate, and formic acid was achieved with a simple electrolyte composed by 2 mM potassium dichromate as the carrier solution and background absorbance provider and 0.05 mM tetraethylenepentamine (TEPA) as electro-osmotic flow suppressor (pH 4.00). Injection was performed hydrostatically by elevating the sample at 10 cm for 10 s. The running voltage was -27 kV at 25 degrees C. Indirect UV absorption detection was achieved at 254 nm. The detection limit was in the range between 0.03 and 20 mg/kg, and the quantification limits ranged from 1.52 to 20.6 mg/kg. The calibration graphs were linear in the concentration range from the quantification limit to at least 2.5 g/kg for chloride, 0.25 g/kg for nitrate, 0.75 g/kg for sulfate, 1.50 g/kg for phosphate, and 0.75 g/kg for formic acid. Precision data in the honey samples analyzed showed repeatability and reproducibility relative standard deviations lower than 1.4 and 2.4% for migration time and lower than 1.8 and 4.3% for anion content, respectively. Recoveries of anions in honey samples analyzed ranged from 94.4 to 99.8%. Ten honey samples were analyzed to test the proposed method. Mean contents of 260.5, 3.93, 60.5, 139.4, and 209.3 mg/kg were found, respectively, for chloride, nitrate, sulfate, phosphate, and formic acid in analyzed honeys. These results agreed with literature data.

The determination of low molecular weight organic acids in grape juices and wines is important because of their influence in organoleptic properties (flavour, colour and aroma) and in the stability and microbiologic control of these beverages. The analysis of these acids allows to check the process of maturation of grapes and to control the evolution of the acidity of wines during the several stages of their elaboration process (alcoholic fermentation, malolactic fermentation, aging process, etc). In this work, a comprehensive review of the analytical methods developed for the determination of short-chain organic acids in grape juices and wines has been shown. This review included non-enzymatic spectrophotometric, enzymatic, chromatographic and electrophoretic methods. Details of the chromatographic and electrophoretic conditions of these methods have been collected in tables.

A capillary electrophoresis system for the simultaneous determination of cations in honey samples has been developed. The complete separation and quantification of K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+, which represent more than 99% of the total content of cations in honey, can be achieved in 4 min with only a dilution and filtration of the honey sample. Electrolyte solution was composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). The running voltage was + 25 kV at 25 degree C. Indirect UV detection was achieved at 185 nm. Under the optimum conditions the detection limits ranged from 0.02 to 48.2 mg/kg and the quantification limits have ranged from 0.41 to 48.7 mg/kg. Precision data in honey samples analysed have shown repeatability and reproducibility RSD (%) lower than 2.84 and 6.62%, respectively. Recoveries of cations in honey samples analysed have ranged from 88.5 to 101.8%. These cations were identified by their relative migration times with regard to Ba2+ migration time used as reference standard and they were quantified by using an external standard calibration. Twenty-five honey samples were analysed to test the proposed method. Mean contents of 1.22 x 10(3), 93, 85, 54, 11, 1.9 and 2.3 mg/kg were found, respectively, for K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+ cations in analysed honeys. These results were similar than the obtained by other authors.

The total contents of maleic and fumaric acids (cis and trans-2-butenedioic acids, respectively) were quantified by a high performance liquid chromatography method, in 50 floral honeys of Galicia (north-western Spain). Honey pH, activity coefficients, dissociation constants of the acids (K1 and K2), and the molar concentrations of the forms of maleic and fumaric acids naturally present in honey ([AH2], [AH−] and [A2−]) have been calculated for the first time. The contents of maleic and fumaric acids can be determined either as total maleic and fumaric acids ([AH2]) or as total maleate and fumarate ([A2−]), but there are other forms of these acids in honey. Therefore the calculation of the forms of the maleic and fumaric acids present would illuminate their origin at honey pH. The predominant acid form depends on honey pH value. Maleic acid was quantifiable in 44 honeys. The [AH−] form was found as a major component in all samples. Fumaric acid was quantifiable in 49 honeys. The [A2−] form was found as a major component in most honeys (28 samples) and the [AH−] was predominant in 21 samples. No honey analysed had a [AH2] form as predominant. Although maleic and fumaric acids have the same molecular weight and they are both dicarboxylic acids, their pH relationships differ. The relationships between maleic and fumaric acids and honey pH and between the total content of maleic and fumaric acids and their forms in honey have been calculated.

A rapid high-performance liquid chromatographic method for the determination of organic acids in honey is reported. Malic, maleic, citric, succinic and fumaric acids were identified and quantified in 15 min. First time repeatibility, reproducibility and recoveries were determined out for these acids in honey samples. Maleic acid was also quantified for first time by a chromatographic method. The organic acids were removed from honey by using a solid-phase extraction procedure with anion-exchange cartridges. Previously, the solution of honey was adjusted to pH 10.50 with 0.1 M NaOH and stirred for 15 min at room temperature. Then, this solution was adjusted to pH 5.00 with 0.1 M H2SO4. This procedure was carried out to avoid interferences in the baseline. The chromatographic separation was achieved with only one Spherisorb ODS-2 S5 column thermostated at 25 degrees C. Metaphosphoric acid (pH 2.20) was used as mobile phase at a flow-rate of 0.7 ml/min. Organic acids were detected with a UV-vis detector (215 nm). The precision results showed that the relative standard deviations of the repeatability and reproducibility were < or =3.20% and < or =4.86%, respectively. The recoveries of the organic acids ranged from 62.9 to 99.4%. Under optimum conditions the detection limits ranged from 0.0064 to 7.57 mg/kg and the quantification limits ranged from 0.025 to 10.93 mg/kg.

A rapid and reliable capillary zone electrophoresis method for the determination of inorganic cations was developed. The complete separation of K + , Ba 2+ , Ca 2+ , Na + , Mg 2+ , Mn 2+ , Ni 2+ , Cd 2+ , Li + and Cu 2+ can be achieved in 4 min with a simple electrolyte composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). Injection was performed hydrostatically by elevating the sample at 10 cm for 30 s. The running voltage was +25 kV at room temperature. Indirect UV-absorption detection was achieved at 185 nm. The detection limit was in the range between 0.06 mg/l (Mg 2+ ) and 0.57 mg/l (K + ) and the quantification limits ranged from 0.10 mg/l (Ni 2+ ) to 0.80 mg/l (Cu 2+ ). The calibration graphs were linear in the concentration range from the quantification limit till at least 1 g/l in K + , 10 mg/l in Ba 2+ , Ca 2+ , Mg 2+ , Mn 2+ , Ni 2+ and Cd 2+ , 40 mg/l in Na + and 12 mg/l in Li + and Cu 2+ . The repeatability, intraday and interday analysis were ≤1.55% and ≤3.64% for migration time and ≤3.38% and ≤3.63% for peak area. The method developed has been applied to several beverage samples with only a simple dilution and filtration treatment of the sample. The proposed method is simple, fast, cheap and it is achieved with common products in either laboratory. For these reasons, it is a very useful method for routine analysis.

A solid-phase extraction procedure was applied to remove organic acids from honey. Malic, maleic, citric, succinic and fumaric acids were isolated with an anion-exchange cartridge. The different parameters which affected the extraction procedure were studied and optimised to establish the optimal conditions for maximum recovery of organic acids and minimum extraction of interferences. The optimised procedure used a cartridge which was activated with 10 ml of 0.1 M sodium hydroxide solution (percolation rate 3 ml/min). A 10 ml volume of honey solution was passed at a flow-rate of 0.5 ml/min. The cartridge was washed with 10 ml of water (3 ml/min) and organic acids were eluted with 4 ml of 0.1 M sulfuric acid (0.5 ml/min). This solution was injected directly into the chromatograph. When this procedure was carried out on standard solutions of organic acids, recoveries between 99.2 and 103.4% were found. If this procedure was applied to honey samples these recoveries were also satisfactory and ranged from 62.9 to 99.4%.