# Effect of Production Technique on Pilsner-Style Non-Alcoholic Beer (NAB) Chemistry and Flavor
## 2 Materials and Methods
### Volatile Beer Analyses  
For the analysis of volatiles, either industry standard methods, published peer-reviewed methods, or modified published methods were applied. The chemicals and instruments for the industry standard methods as well as the published methods were the same as previously reported in the associated citation, while for the modified methods utilized, more detailed information is provided below. For all of the analyses, duplicate measurements were performed and the mean values are reported herein. As mentioned above, the measurement precision is reported as the relative standard deviation (%RSD) calculated by performing a 6-fold repeat analysis (Tables S2-S4). Dimethyl sulfide (DMS) was measured using HS-GC with a pulsed flame photometric detector (PFPD) operated under conditions outlined in MEBAK 2.23.1.1 [23]. Hop-derived aroma compounds were analyzed by headspace solid-phase microextraction (HS-SPME) GC-MS/MS according to the method described previously by [PERSON], et al. [24]. Aldehydes were analyzed by HS-SPME and on-fiber derivatization (OFD) GC-MS/MS as published by [PERSON] et al. [25]. The concentrations of acetaldehyde, higher alcohols, and acetate esters were determined by static headspace gas chromatography with flame ionization detection (HS-GC-FID) according to the method EBC 9.39 [22]. The GC-FID instrument used was a Shimadzu GC-2010 (Shimadzu Corp., Kyoto, Japan) equipped with a DB-Wax (60 m \(\times\) 0.32 mm \(\times\) 0.5 \(\upmu\)m film thickness) from Agilent (Santa Clara, CA, USA). Quantification of higher alcohols and esters was reached using butan-1-ol and phenol as internal standards added at a concentration of 15 mg/L. Phenol was used to quantify phenyl ethanol and phenylethyl acetate, while the other volatiles were quantified using butan-1-ol. The calibration ranges of the compounds differed and were as follows: acetaldehyde (1-75 mg/L), ethyl acetate (0.5-75 mg/L), propanol (0.5-75 mg/L), isobutanol (1-75 mg/L), isoamyl acetate (0.5-10 mg/L), 2-methyl-1-butanol (1-75 mg/L), 3-methyl-1-butanol (1-100 mg/L), phenyl ethanol (1-75 mg/L), and phenylethyl acetate (0.1-5 mg/L) The lowest concentration of the calibration range was defined as limit of quantification for each compound. Ethyl esters (i.e., ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, and ethyl dodecanoate) were determined by headspace-solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). The GC-MS system used was a Shimadzu GC-2010 interfaced with a MS-QP2010 Plus (Shimadzu Corp., Kyoto, Japan)equipped with a Gerstel MPS 2 XL auto sampler (Gerstel, Muhlheim an der Ruhr, Germany) for automated HS-SPME sampling. Esters were extracted from 2 mL liquid sample using a 50/30 \(\upmu\)m DVB/CAR/PDMS fiber (Supelco, St. Louis, MO, USA), the column used for chromatographic separation was an HP-5 MS UI column (30 m \(\times\) 0.25 mm i.d. \(\times\) 0.25 \(\upmu\)m film thickness from Agilent (Santa Clara, CA, USA)). The extraction and GC parameters used are described by [PERSON], [PERSON], [PERSON], and [PERSON] [24]. Briefly, the mass spectrometer was operated in selected ion monitoring (SIM) mode, using the following qualifier and quantifier ions: ethyl butyrate (m/z 71 and m/z 60), ethyl hexanoate (m/z 88 and m/z 101), ethyl octanoate (m/z 88 and m/z 127), ethyl decanoate (m/z 88 and m/z 73), and ethyl dodecanoate (m/z 88 and m/z 101). The calibration range for all ethyl esters ranged from 5 \(\upmu\)g/L to 1000 \(\upmu\)g/L. The lowest concentration of the calibration range (5 \(\upmu\)g/L) was defined as the limit of quantification. Isotopically labeled d5-ethyl hexanoate (m/z 93 and m/z 106), was used as an internal standard at a concentration of 100 \(\upmu\)g/L. Data evaluation was done using the GC-MS solution software Version 4.45 SP1 (Shimadzu Corp., Kyoto, Japan).