# What's in Your Fruit Juice?--Rapid Quality Screening Based on Infrared (FT-IR) Spectroscopy
## 2 Materials and Methods
### Reference Analysis  
The analysis of sugars (sucrose, glucose, and fructose) was conducted through high-performance liquid chromatography (HPLC). The juice samples underwent filtration using non-sterile 0.45 \(\upmu\)m pore size filters (Phenomenex(r), Torrance, CA, USA) into 2 mL number colored HPLC vials. Subsequently, the vials were stored at -18 \({}^{\circ}\)C until the analysis. For the sugar analysis, a Shimadzu Prominence UFLC (Shimadzu, Columbia, MD, USA) system was utilized, featuring dual LC-6 AD pumps, an SIL-20 AHT auto-sampler, a CTO-20A column oven, and an RID-10A refractive index detector. The elution process involved passing the sugars through a 7.8 mm ID \(\times\) 300 mm ion exclusion column (Rezex RCM-Monosaccharide column, Ca+2, 8 \(\upmu\)m, Torrance, CA, USA) at a constant flow rate of 1 mL/min and a temperature of 80 \({}^{\circ}\)C, with the entire run lasting 30 min. The total sugar content in the analyzed FJ samples was determined by summing all three individual sugars. LC Solutions software version 3.0 (Shimadzu Scientific Instruments Inc., Columbia, MD, USA) was used to collect and analyze the chromatograms.  
The measurement of soluble solids (\({}^{\circ}\)Brix) was conducted at 20 \({}^{\circ}\)C using a digital refractometer (Atago, Model RX-5000i, Bellevue, WA, USA), presenting results in percentage terms. The assessment of titratable acidity in the FJ samples was conducted using the official method specified by AOCS, employing an automatic titrator (Easy Plus Titration, Mettler Toledo, Greifensee, Switzerland).  
Ascorbic and citric acids were quantified in juice samples using an Agilent HPLC system (1100 Series, Agilent Technologies, Santa Clara, CA, USA) equipped with a G1311A quaternary pump, G1322A degasser, G1313 ALS autosampler, G1316A column compartment, and G1315B diode array detector. After passing through non-sterile 0.45 \(\upmu\)m filters into amber-colored 2 mL HPLC vials, 100 \(\upmu\)L of TCEP was added and left overnight, acting as a reducing agent to convert oxidized dehydroascorbic acid back to ascorbic acid [33]. Adding a reducing agent enhanced the stability of vitamin C during the extraction and storage period in the autosampler during the HPLC analysis. The stability of ascorbic acid in sample extracts demonstrated stabilization for a minimum of 96 h. Additionally, the addition of TCEP as a reducing agent with an overnight incubation has proven to not induce any interference with the chromatographic system [33]. A total of 10 \(\upmu\)L of a sample was injected into a stainless-steel HPLC-grade acidified water (pH 2.2, acidified with H\({}_{2}\)SO\({}_{4}\)) facilitated isocratic separation at a flow rate of 0.8 mL/min for 30 min on a stainless-steel, 4.6 mm ID \(\times\) 150 mm Prevail organic acid column with 5 \(\upmu\)m spherical packing (Hichrom Limited, Leicestershire, UK). Chemstation software (v B.04.03 Agilent, Santa Clara, CA, USA) was utilized for capturing and analyzing the chromatograms. Ascorbic and citric acids were discerned by matching their peak retention times with standards of pure ascorbic and citric acids. All the reference analyses were performed in duplicate.