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MBAA TQ vol. 54, no. 2 • 2017 • pp. 72–79
PEER-REVIEWED PAPER
Comparison Between DTNB and p-Rosaniline
Methods to Quantify Total SO2 in Beer
Maria E. Moutsoglou
Research and Development, Sierra Nevada Brewing Company, Chico, CA, U.S.A.
ABSTRACT
Food products containing sulfites (SO2) above 10 mg/L are legally
required to have “contains sulfites” clearly displayed on packaging.
Accurately quantifying SO2 in beer is necessary to ensure sulfites are
below the 10 mg/L limit. One common method of quantifying total
SO2 (free and complexed) is using a segmented flow analyzer (SFA)
with p-rosaniline (PRA) as a chromophore. The accuracy of this
method was assessed in comparison to a discrete flow analyzer (DFA)
using 5-5′-dithio-bis(2-nitrobenzoic acid) (DTNB) as a chromophore.
SO2 was supplemented into beer and was quantified using the DFA
and SFA. Each method accurately quantified the amount of supple-
mented SO2; however, there was a ≈3.6 mg/L difference in total SO2.
To determine if this difference was due to the ability of the dyes to
bind complexed SO2, SO2 was supplemented in a buffered solution
and in lager and ale beers (to measure free SO2), and in an acetalde-
Sulfur dioxide (called sulfites, bisulfites, or SO2) in beer
contributes to flavor stability and improved shelf life. Sulfites
act as antioxidants, preventing the formation of aldehydes,
and can also form adducts with carbonyl compounds to en-
hance flavor stability. Free sulfites (i.e., not bound to carbon-
yls) also have antimicrobial properties (1,2). Sulfites can also
form reversible and nonreversible complexes with molecules
in solution, and this is known as bound sulfite. The total
sulfites in solution are the sum of the free and bound sulfites.
Unfortunately, sulfites at certain concentrations can trigger al-
lergic reactions in sensitive people. Symptoms can be minor,
such as a rash, but sulfites can also cause life-threatening asthma
and anaphylaxis (3). Both the United States and European
Union have mandated the labeling of alcoholic beverages
containing sulfites above 10 mg/L with “contains sulfites” or
“contains (a) sulfating agent(s)” (1). This number is measured
as total sulfites.
Sulfites are produced during fermentation but may also re-
sult from priming or finings additions. Lager yeast strains can
produce up to ≈30 mg of SO2/L during sulfur-containing amino
acid synthesis (i.e., cysteine and methionine), and some of this
may carry over as sulfite into the finished product (2). At con-
centrations greater than 20 mg/L, SO2 may cause off-flavors in
beer (2). Sulfite production by yeast is influenced primarily by
factors affecting yeast health (vitality), such as pitching rate,
E-mail: maria.moutsoglou@sierranevada.com
http://dx.doi.org/10.1094/TQ-54-2-0314-01
© 2017 Master Brewers Association of the Americas
72
hyde-saturated buffered solution and lager beer (to measure com-
plexed SO2). Although PRA was consistently less accurate than
DTNB in quantifying supplemented bound and free SO2 in both a
buffered solution and beer, both methods remained within approxi-
mately ±3 mg/L of the supplemented SO2 target. In unsupplemented
beer, however, an up to 5.6 mg/L difference was observed in total SO2
between the two methods, in which PRA consistently quantified lower
total SO2 than DTNB. This difference corresponded to a maximum
observed percent difference of 127.3% for lager beers. Lagers from
other breweries were tested, and the percent difference between
DTNB- and PRA-derived SO2 decreased linearly as the concentration
of SO2 increased. This suggests PRA has a larger limit of detection for
SO2 in beer produced during fermentation than DTNB, and it should
not be used to quantify total SO2 in beer.
fermentation temperature, wort pH, wort strength, fermenter
pressure, trub (proteinaceous) content, and wort aeration (2).
High wort pH, low wort oxygenation, and low yeast pitching
rates increase the production of sulfites (2). In a beer medium,
in which the pH is typically acidic (pH ≈ 3.8–4.4), free sulfites
are found in the form of bisulfite ions (HSO3–) (1). The half-
life of SO2 in packaged beer stored at 25°C is around 3–6
months (4), and it is influenced by reactions with oxygen,
carbonyls, and other beer components (2). Increased storage
temperature decreases the half-life of sulfites (4). The accurate
quantification of sulfites is particularly important in lager
beers, which typically contain more sulfites than ales.
Two colorimetric methods appropriate for a brewery setting
(ease of use and accuracy) were chosen to compare: the p-rosan-
iline (PRA) method and the 5-5′-dithiobis(2-nitrobenzoic acid)
(DTNB) method. PRA only binds to free sulfites and requires
heat or other elements to free sulfite from its potential bonded
component. The sulfite binds to PRA in the presence of hydro-
chloric acid and formaldehyde reagents, and the sulfite–PRA
compound strongly absorbs at 550 nm (5). DTNB is an or-
ganic disulfide (RSSR) that reacts with sulfite to produce thio-
nitrobenzoate (TNB, RS–), which strongly absorbs at 420 nm (6).
Li and Zhao assessed colorimetric methods for sulfites in
beer and have shown that DTNB and PRA are practical for
quantifying total sulfites in beers up to 5 mg/L (7); however,
sulfite concentrations in lagers can be greater than 5 mg/L.
Therefore, validating these methods at higher concentrations is
important not only for validating the organoleptic quality of
beer but also for ensuring the legality of the shipped product.
A segmented flow analyzer (SFA) (Astoria segmented ana-
lyzer, Astoria-Pacific, Clackamas, OR, U.S.A.) used in Sierra
Methods to Quantify Total Sulfur Dioxide in Beer
Nevada Brewing Company (SNBC) quality assurance uses
PRA to quantify SO2; unfortunately, this method requires
harmful reagents and is time-consuming to run. A total SO2
method was developed with DTNB on a discrete flow analyzer
(DFA) (Astoria discrete analyzer, Astoria-Pacific). A study
comparing the two instruments’ methods was conducted to
determine the accuracy and precision of measuring total sul-
fites in both stabilized buffer and in beer, up to a total sulfite
concentration of 20 mg/L. The results of this study suggest
that DTNB is the more accurate method for quantifying sul-
fites in beers.
Methods
Total SO2 Differences in Lager Beers
Beer samples were filtered through Whatman number 995
filter paper. Free SO2 (from sodium metabisulfite, Na2S2O5) at
0, 1, 5, and 10 mg/L was added to packaged lager A from three
separate lager fermentations. Total SO2 was determined using
DTNB in a DFA (Astoria discrete analyzer). SO2 stock (50
mg/L) was made by dissolving sodium metabisulfite in stabi-
lizing buffer (SB; 38 mM sodium phosphate dibasic [Na2HPO4]
and 10 mM D-mannitol [C6H14O6]). The DFA used the 50
mg/L stock to generate a calibration curve with 0, 1, 5, 10, and
20 mg/L of SO2. Samples and standards were mixed with Tris
buffer (17 mM Tris, 33 mM Tris-HCl, pH 7.8). A stock solu-
tion of DTNB was prepared by dissolving DTNB in 10% v/v
200 proof ethanol first and then adding 90% v/v double-dis-
tilled H2O. DTNB was added to the sample, and the solutions
were incubated at 37°C for 15 min. The absorbance at 405 nm
was used to generate a calibration curve fit using second-order
polynomial regression (typically R2 > 0.99). Standards were
prepared and used within three days.
SO2 was quantified using the SFA (Astoria segmented ana-
lyzer) with PRA as a colorimetric agent. Standards for a calibra-
tion curve were prepared at 0.5, 1, 2, 5, and 10 mg/L of SO2
(from sodium metabisulfite) in SB. Samples were passed
through a heating block at 32.2°C to denature any bound SO2.
The calibration curve was fit using second-order polynomial
regression (typically R2 > 0.999) to calculate sample SO2
concentrations in milligrams per liter. The reagents used by the
segmented analyzer were 1N hydrochloric acid with 0.05% v/v
Aerosol-22 surfactant, 0.05N sulfuric acid with 0.025% v/v
Ethomid ethoxylated amide, and 0.2% v/v formaldehyde
reagent.
Figure 1. A, Average and standard deviation (error bars, n = 3 trials) of total SO2 values in lager A quantified using 5,5′-dithio-bis(2-nitrobenzoic
acid) (DTNB) (blue) or p-rosaniline (PRA) (red). Data were fitted using linear regression, for which the slopes of the discrete and segmented fits
were –0.065 and –0.066, respectively. B, The average deviation from expected (measured increase in SO2 – supplemented SO2) for the DTNB and
PRA. Data were fitted using linear regression. Coloring is retained from A.
MBAA TQ vol. 54, no. 2 • 2017 73
Bound and Free SO2 Differences in SB
SO2 samples were prepared in SB or in SB supplemented
with 100 mg/L of acetaldehyde. SO2 standard (50 mg/L) was
prepared in SB. Samples were prepared and run on the DFA
and SFA on the same day. SO2 stock solutions and standards
were used within three days of preparation. A check standard
made from sodium sulfite (Na2SO3) was included in both in-
struments, and data were rejected if the check standard failed.
Bound and Free SO2 Differences in SNBC Beer
SO2 was added to filtered beer or beer supplemented with
100 mg/L of acetaldehyde and was run on the same day on the
DFA and SFA. A stock solution of SO2 (1 g/L) was made in
each and was diluted in beer to produce the samples. A check
standard made from sodium sulfite was included in both in-
struments, and data were rejected if the check standard failed.
SO2 Quantification in Other Breweries’ Lagers
Beers from other breweries were purchased on the date of the
experiment. Beer was filtered and run on the DFA and SFA.
Data Analysis
Linear regression and statistical analyses were performed
using GraphPad Prism 6.0h (GraphPad Software, La Jolla, CA,
U.S.A.). The Student’s t test was used to determine statistical
significance when P ≤ 0.05. Percent difference between two
values (V1, V2) was determined as follows:
V1 − V2
% difference = ×100
 V1 + V2 
 2 
 
Results and Discussion
Initial Quantification of SO2 Supplemented in Lager
Beer Showed Methods-Based Differences in SO2
Produced During Fermentation
To assess the precision and accuracy of the PRA and DTNB
methods for quantifying total SO2, SO2 was supplemented into
SNBC lager A beer (n = 3 distinct beers packaged from differ-
ent fermenters). A nonsupplemented sample and samples sup-
plemented with 1, 5, and 10 mg/L of SO2 were analyzed using
both methods. The absorption values were converted into SO2
from a calibration curve using software provided with each
74 MBAA TQ vol. 54, no. 2 • 2017 Methods to Quantify Total Sulfur Dioxide in Beer

instrument. The DFA software prepares standard curve sam-
ples from a stock standard of SO2, whereas the SFA requires
the user to make standard solutions.
Figure 1A shows the total SO2 in each sample using DTNB
or PRA. Because the concentration of SO2 should increase
linearly in the supplemented samples, linear regression was
used to compare the accuracy of each method to quantify sup-
plemented SO2. The slopes of the PRA and DTNB lines were
not significantly different (P > 0.05), suggesting a good corre-
lation between both instruments’ ability to quantify supple-
mented SO2. Tables 1 and 2 show measured SO2, the expected
and measured increases from supplemented SO2, and the dif-
ference between expected and measured values from the DTNB
and PRA methods, respectively. Figure 1B shows the deviation
from the expected concentration of SO2, which is defined as
the difference in measured SO2 from supplemented SO2. As
the concentration of supplemented SO2 increased, both meth-
ods underestimated the supplemented SO2 concentration, for
which the underestimation increased as supplemented SO2
increased.
Although both methods could quantify the supplemented
SO2 in lager beer, there was a significant difference (P ≤ 0.05)
in the total SO2 in solution between the two methods. SO2
quantified using DTNB was on average 3.6 ± 0.1 mg/L higher

Table 1. Average total SO2 (mg/L) ± standard deviation from n = 3 lager beer samples quantified using DTNBa
Sample ID Measured Supplemented Measured increase Deviation from expected increase
A 8.3 ± 0.8 0.0 0.0 ± 0.0 0.0 ± 0.0
B 9.1 ± 0.6 1.0 0.8 ± 0.3 –0.2 ± 0.3
C 12.8 ± 0.3 5.0 4.6 ± 0.5 –0.4 ± 0.5
D 17.4 ± 0.5 10.0 9.1 ± 0.4 –0.9 ± 0.4
a Measured increase is calculated as measuredi – measuredA, where i is the sample ID. Deviation from expected is the measured increase in SO2 – supple-
mented SO2. DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid).

Table 2. Average total SO2 (mg/L) ± standard deviation from n = 3 lager beers quantified using PRAa
Sample ID Measured Supplemented Measured increase Deviation from expected increase
A 4.7 ± 0.8 0.0 0.0 ± 0.0 0.0 ± 0.0
B 5.7 ± 0.8 1.0 1.0 ± 0.1 0.0 ± 0.1
C 9.1 ± 0.8 5.0 4.5 ± 0.3 –0.5 ± 0.3
D 13.9 ± 0.7 10.0 9.2 ± 0.4 –0.8 ± 0.4
a Measured increase is calculated as measuredi – measuredA, where i is the sample ID. Deviation from expected is the measured increase in SO2 – supple-
mented SO2. PRA = p-rosaniline.

Table 3. Average total SO2 (mg/L) in stabilizing buffer ± standard deviation (SD) (n = 2 measurement) quantified using PRA or DTNBa
PRA DTNB
Standard SO2 (mg/L) Average ± SD Deviation from standard Average ± SD Deviation from standard
0 0.10 ± 0.00 0.10 0.08 ± 0.04 0.08
2 1.85 ± 0.07 –0.15 1.96 ± 0.00 –0.04
3 2.75 ± 0.07 –0.25 2.94 ± 0.07 –0.06
4 4.05 ± 0.04 0.05 3.71 ± 0.29 –0.29
5 4.90 ± 0.00 –0.10 4.94 ± 0.29 –0.06
10 9.65 ± 0.07 –0.35 10.51 ± 0.29 0.51
26 23.95 ± 0.07 –2.05 26.22 ± 0.19 0.22
a The deviation from the standard concentration is shown (measured SO2 – standard SO2). PRA = p-rosaniline; and DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid).

Figure 2. A, Total SO2 supplemented in stabilizing buffer quantified using 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (blue) or p-rosaniline
(PRA) (red). Error bars represent standard deviation from n = 2 measurements. Intersecting grey lines show the expected concentration of SO2. B,
Deviation from expected (measured SO2 – added SO2) for SO2 quantified using DTNB or PRA. Coloring retained from A.
Methods to Quantify Total Sulfur Dioxide in Beer MBAA TQ vol. 54, no. 2 • 2017 75

than with PRA. This suggests each method quantifies SO2 dif-
ferently in beer, but only SO2 produced during fermentation.
Quantifying Free SO2 in a Buffered Solution
To assess SO2 quantification with DTNB and PRA in a buff-
ered solution, SO2 was supplemented into SB (38 mM sodium
phosphate dibasic [Na2HPO4] and 10 mM D-mannitol [C6H14O6])
(Table 3, Fig. 2). Both methods showed minor deviations from
expected values as the concentration of SO2 increased up to 10
mg/L. At 20 mg/L, the PRA-derived SO2 concentrations devi-
ated from the expected value by –2.05 mg/L, compared with
0.22 mg/L when using DTNB; however, 26 mg/L was greater
than the maximum calibration curve standard (10 mg/L of
SO2) used in the PRA method, and this likely explains the
larger deviation.
Quantifying Free SO2 in Beer
To determine if DTNB and PRA could comparably quantify
SO2 in both an ale and lager beer supplemented with free SO2,
SO2 was added to a packaged ale (SNBC ale A) and lager
(SNBC lager B). Figure 3A and C shows SO2 quantified for
supplemented ale A and lager B, respectively; Figure 3B and D
shows the deviation from expected (measured increase in SO2
– supplemented SO2) in ale A and lager B, respectively. For ale
A, the average deviation from expected using PRA and DTNB
was –2.46 and –1.24 mg/L, respectively, suggesting that
DTNB more accurately quantified SO2 in ale beer. For lager B,
the average deviation from expected was –0.80 and –0.45
mg/L using PRA and DTNB, respectively, showing a similar
trend of accuracy as in ale A, although the average deviation
was smaller for both instruments in lager B than in ale A.
There may be some interfering agent in ale beer that increases
variability in quantifying SO2. However, DTNB consistently
quantified SO2 more accurately than PRA. Data from Figure 3
are shown in Tables 4–7.
Comparing the slopes from the linear regression fits in ale
A, DTNB and PRA data had significantly different slopes (P ≤
0.05), although the slopes were not significantly different in
lager B. This suggests that different beer solution environment
affects the way each instrument can quantify SO2.
Quantifying Bound SO2 in a Buffered Solution
To determine how each method quantified bound SO2 in a
buffered solution, SO2 was supplemented into SB (as free SO2)
and into SB in the presence of saturating (100 mg/L) acetalde-
hyde (as bound SO2), and it was quantified using DTNB and
PRA (Fig. 4). Data were fitted using linear regression, and the
slopes of the lines for SO2 in the presence and absence of acet-
aldehyde quantified using either DTNB or PRA were not sig-
nificantly different from each other (P > 0.05). The average
deviation from the expected increase in bound SO2 using PRA
and DTNB was 1.2 and 0.8 mg/L, respectively. The similarity
in the slope and the deviation from expected suggests that both
methods could comparably quantify bound SO2 (Tables 8 and
9) in a buffered solution. The deviation from expected in-
creased as the concentration of supplemented SO2 increased.
Quantifying Bound SO2 in Lager Beer
To determine the capability of each method to quantify bound
SO2 in a lager beer, SO2 was supplemented into lager B (as
free SO2) and into lager B in the presence of 100 mg/L of acet-
aldehyde (as bound SO2), and it was quantified using DTNB

Figure 3. A, SO2 measured in ale A using 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (blue) or p-rosaniline (PRA) (red) with respect to SO2
added. Data were fitted using linear regression (dotted lines), for which fits for DTNB and PRA data had R2 = 0.9951 and 0.9586, respectively. B,
Deviation from expected (measured increase in SO2 – supplemented SO2) for samples in A. C, SO2 measured in lager B using DTNB (blue) and
PRA (red) with respect to free SO2 added. Data were fitted using linear regression (dotted lines), for which fits for DTNB and PRA data had R2 =
0.9982 and 0.9996, respectively. D, Deviation from expected for samples in C. A consistent coloring scheme is used in A–D. n = 1 measurement.
76 MBAA TQ vol. 54, no. 2 • 2017 Methods to Quantify Total Sulfur Dioxide in Beer

and PRA (Fig. 5). Data were fitted using linear regression, and suggesting the beers may have been between four and eight
the slopes of the lines for SO2 in the presence and absence of months old. Imported lager 1 had a “drink by” date eight
acetaldehyde quantified using either DTNB or PRA were not months earlier than the date of the experiment, suggesting the
significantly different from each other (P > 0.05). The average beer may have been between 12 and 18 months old. SNBC
deviation from the expected increase in bound SO2 for both lager B and lager A (glass and can) produced eight months
PRA and DTNB was –1.1 and 0.1 mg/L, respectively, suggest- before the experiment date were used as comparably aged
ing that DTNB could more accurately quantify bound SO2 in samples to the outside brand lagers.
lager beer than PRA (Tables 10 and 11).
The concentration of SO2 measured for lager B not supple-
mented with SO2 or acetaldehyde was 7.2 and 1.6 mg/L using
DTNB and PRA, respectively. Because DTNB and PRA ap-
pear to similarly quantify both bound and free SO2 in SB,
some component in the beer may alter the ability of DTNB or
PRA to bind to SO2.
Quantifying SO2 in Non-SNBC Lagers
To determine if other breweries’ lagers show similar dissimi-
larities in quantified SO2 using PRA and DTNB, lagers from
10 different breweries were analyzed. SNBC lager A packaged
in 2007 (aged around nine years) was used as a negative con-
trol; if some component of the lager solution were inflating
total SO2 (in the case with DTNB), then PRA should show
around zero SO2, and DTNB would show some non-zero value.
At nine years past production date, all SO2 should have been Figure 4. SO2 measured in stabilizing buffer in the presence (solid line)
completely oxidized. Most outside brand lagers were pur- and absence (dotted line) of 100 mg/L of acetaldehyde using 5,5′-dithio-
chased from a local grocery retailer on the day of the experi- bis(2-nitrobenzoic acid) (DTNB) (blue) or p-rosaniline (PRA) (red).
ment, with “drink by” dates ranging one to five months later, Data were fitted using linear regression, for which all R2 ≥ 0.999.

Table 4. Average SO2 ± standard deviation for ale A and for ale A supplemented with SO2 using DTNB (n = 2 measurements)a
Supplemented SO2 (mg/L) Measured SO2 (mg/L) Measured SO2 increase (mg/L) Deviation from expected increase (mg/L)
0 1.18 ± 0.21 0.00 0.00
4 4.07 ± 0.07 2.88 –1.12
5 5.46 ± 0.14 4.27 –0.73
10 9.32 ± 0.21 8.14 –1.86
a DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid).

Table 5. Average SO2 ± standard deviation for ale A and for ale A supplemented with SO2 using PRA (n = 2 measurements)a
Supplemented SO2 (mg/L) Measured SO2 (mg/L) Measured SO2 increase (mg/L) Deviation from expected increase (mg/L)
0 0.25 ± 0.07 0.00 0.00
4 1.85 ± 0.07 1.60 –2.40
5 3.05 ± 0.07 2.80 –2.20
10 7.45 ± 0.07 7.20 –2.80
a PRA = p-rosaniline.

Table 6. Average SO2 ± standard deviation for lager B and for lager B supplemented with SO2 using DTNB (n = 2 measurements)a
Supplemented SO2 (mg/L) Measured SO2 (mg/L) Measured SO2 increase (mg/L) Deviation from expected increase (mg/L)
0 11.59 ± 0.00 0.00 0.00
4 15.55 ± 0.28 3.97 –0.03
5 16.22 ± 0.49 4.64 –0.37
10 20.65 ± 0.21 9.06 –0.94
a DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid).

Table 7. Average SO2 ± standard deviation for lager B and for lager B supplemented with SO2 using PRA (n = 2 measurements)a
Supplemented SO2 (mg/L) Measured SO2 (mg/L) Measured SO2 increase (mg/L) Deviation from expected increase (mg/L)
0 7.80 ± 0.00 0.00 0.00
4 11.40 ± 0.14 3.60 –0.40
5 12.10 ± 0.00 4.30 –0.70
10 16.50 ± 0.00 8.70 –1.30
a PRA = p-rosaniline.
Methods to Quantify Total Sulfur Dioxide in Beer MBAA TQ vol. 54, no. 2 • 2017 77

Table 12 and Figure 6 show SO2 quantified using DTNB and
PRA, with the percent difference also shown (Table 12). Im-
ported lagers 1–4 may have been shipped from the manufac-
turer with elevated levels of SO2 to promote beer stability dur-
ing transport, because all four beers had higher levels of SO2
than some domestic and craft brands. Domestic lagers 1 and 2
had SO2 concentrations higher than most of the craft lagers
(CL 1–4) and were all packaged in aluminum cans, which
have been shown to prevent the ingress of O2 into packaged
product, therefore potentially preserving sulfites present in the
beer (8).
Data were plotted from lowest SO2 concentration to highest
as determined using PRA. SO2 concentration and percent dif-
ference appear linearly inversely proportional, in which the
greatest percent difference was when SO2 concentrations (from
DTNB) were below 4 mg/L. The SO2 concentration of the
aged lager A (from 2007) was around zero for both instru-
ments, suggesting that components in beer are most likely not
falsely inflating DTNB-derived SO2. Therefore, the limit of
detection of PRA may be at least 8 mg/L, whereas DTNB
could quantify at least below 2 mg/L (as evident from SO2
quantification in ales, Fig. 3A). The sensitivity of DTNB for
SO2 has been shown to be as low as 0.8 mg/L (6).
The decreased sensitivity of PRA is only evident when meas-
uring SO2 produced during brewing or fermentation, because
PRA could accurately quantify SO2 between 2 and 5 mg/L
supplemented into SB and could quantify supplemented SO2 in
beer.
Understanding Differences in SO2 Quantified Using
DTNB or PRA
Although both methods can accurately quantify supple-
mented SO2 in beer, there is a significant difference in the con-
centration of SO2 quantified in unsupplemented beer between
the two methods. One reason may be that sulfites produced
during fermentation in bound form (other than acetaldehyde
adducts) are undetectable using the PRA method. This would
yield falsely lower SO2 concentrations, evident in Figures 1, 3,
5, and 6. Although heat is applied to the sample to denature
adducts, the duration of heating may not be enough to denature
all adducts. Some of the molecules that form adducts are re-
viewed below for their potential to bind with bisulfites in beer.
Carbonyl-bisulfite adducts. Carbonyls form stable adducts
with bisulfite to form hydroxysulfonates (2). Acetaldehyde
hydroxysulfonates are the most abundant, because acetalde-
hyde is generated at higher concentrations than other carbonyls
during fermentation (9). Even when bisulfite is not in excess,
acetaldehyde adducts are readily and rapidly formed, and most
bisulfite adducts in beer are with acetaldehyde (10,11). Be-
cause PRA was able to quantify supplemented SO2 in SB and
beer in the presence of saturating acetaldehyde, most likely
bisulfite-acetaldehyde adducts are not causing a decrease in
signal.
Sugar-bisulfite adducts. Bisulfites can form adducts with
reducing sugars such as glucose to form sugar hydroxysul-
fonates. Sugar hydroxysulfonates are not formed as easily as
carbonyl adducts and are less stable (12).
DTNB reacting with sulfur-containing amino acids. An-
other potential explanation for the concentration differences is
that DTNB may be reacting with some non-bisulfite molecule
Figure 5. SO2 measured in lager B in the presence (solid line) and
absence (dotted line) of 100 mg/L of acetaldehyde using 5,5′-dithio-
bis(2-nitrobenzoic acid) (DTNB) (blue) or p-rosaniline (PRA) (red).
Data were fitted using linear regression, for which all R2 ≥ 0.999.

Table 8. Total SO2 concentrations in stabilizing buffer (SB) or in buffer supplemented with 100 mg/L of acetaldehyde determined using DTNBa
Supplemented SO2 Measured SO2 Measured SO2 increase Deviation from expected
(mg/L) Condition (mg/L) (mg/L) increase (mg/L)
0 SB + 100 mg/L of acetaldehyde 0.3 0.0 0.0
SB 0.2 0.0 0.0
8 SB +100 mg/L of acetaldehyde 9.0 8.7 0.7
SB 8.6 8.3 0.3
20 SB+ 100 mg/L of acetaldehyde 21.9 21.6 1.6
SB 20.8 20.6 0.6
a The deviation from expected is the difference between the measured increase and SO2 added. DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid).

Table 9. Total SO2 concentrations in stabilizing buffer (SB) or in buffer supplemented with 100 mg/L of acetaldehyde determined using PRAa
Supplemented SO2 Measured SO2 Measured SO2 increase Deviation from expected
(mg/L) Condition (mg/L) (mg/L) increase (mg/L)
0 SB + 100 mg/L of acetaldehyde 0.3 0.0 0.0
SB 0.4 0.0 0.0
8 SB +100 mg/L of acetaldehyde 9.0 8.7 0.7
SB 8.7 8.3 0.3
20 SB+ 100 mg/L of acetaldehyde 21.9 21.6 1.6
SB 22.6 22.2 2.2
a The deviation from expected is the difference between the measured increase and SO2 added. PRA = p-rosaniline.
78 MBAA TQ vol. 54, no. 2 • 2017 Methods to Quantify Total Sulfur Dioxide in Beer

in beer, causing a falsely larger SO2 concentration. These mole-

cules would need to contain a reduced or free thiol to react
with DTNB to produce TNB. One potential source of free
thiols is the amino acid cysteine. Typically, the concentration
of amino acids is dependent on the wort composition (13).
However, Lund and Andersen showed that sweet wort con-
tained no detectable thiols, but there were detectable amounts
of reduced or free thiols in beer, suggesting that yeast reduce
oxidized cysteines or secrete cysteines during fermentation (14).
Most of the cysteines in beer are in the oxidized form (≈90%),
potentially in the form of disulfide bridges (15). Sulfites can
cleave these disulfide bonds to form a thiol (R-SH) and an
S-sulfonate (R-SSO3–), which leaves the thiol open to reaction
with DTNB, potentially causing an inflation in signal. Methio-
nine can also be oxidized by sulfites to methionine sulfoxide
(16). Although these amino acids are a potential source of in-
flated signal, the cysteine and methionine in proteins are likely
unavailable for oxidation by sulfites due to steric hindrance. The Figure 6. Total SO2 (left y axis) measured using 5,5′-dithio-bis(2-
reaction temperature (37°C) and pH (7.8) are not high or basic nitrobenzoic acid) (DTNB) (blue) or p-rosaniline (PRA) (red) flow
enough to denature the proteins and present structurally buried analyzers for Sierra Nevada lagers and for lagers from other brewer-
cysteines and methionines to the surface (17). ies. The % difference is shown in black (right y axis).

Table 10. Total SO2 concentrations in lager B or in lager B supplemented with 100 mg/L of acetaldehyde determined using DTNBa
Supplemented SO2 Measured SO2 Measured SO2 increase Deviation from expected
(mg/L) Condition (mg/L) (mg/L) increase (mg/L)
0 SB + 100 mg/L of acetaldehyde 6.8 0.0 0.0
SB 7.2 0.0 0.0
8 SB +100 mg/L of acetaldehyde 14.4 7.6 –0.4
SB 15.0 7.9 –0.2
20 SB+ 100 mg/L of acetaldehyde 27.7 20.9 0.9
SB 27.3 20.2 0.1
a The deviation from expected is the difference between the measured increase and SO2 added. DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid).

Table 11. Total SO2 concentrations in lager B or in lager B supplemented with 100 mg/L of acetaldehyde determined using PRAa
Supplemented SO2 Measured SO2 Measured SO2 increase Deviation from expected
(mg/L) Condition (mg/L) (mg/L) increase (mg/L)
0 SB + 100 mg/L of acetaldehyde 1.4 0.0 0.0
SB 1.6 0.0 0.0
8 SB +100 mg/L of acetaldehyde 9.0 7.6 –0.4
SB 8.9 7.3 –0.7
20 SB+ 100 mg/L of acetaldehyde 20.4 19.0 –1.0
SB 19.5 17.9 –2.1
a The deviation from expected is the difference between the measured increase and SO2 added. PRA = p-rosaniline.

Table 12. Total SO2 (mg/L) measured using DTNB and PRA for SNBC lagers and for lagers from other breweriesa
Origin Beer DTNB PRA % Difference
SNBC Lager A (aged 9 years) 0.1 0.2 –1
Craft CL 1 3.4 0.2 178
Craft CL 2 1.5 0.2 154
SNBC Lager B (aged 8 months) 3.6 0.4 160
Craft CL 3 3.0 0.5 143
SNBC Lager A (aged 8 months) 3.0 1.0 100
Craft CL 4 3.0 1.0 101
SNBC Lager B (can, aged 8 months) 4.3 1.3 107
Imported IL 1 4.2 1.5 95
Domestic DL 1 5.3 1.9 94
Imported IL 2 5.2 2.5 69
Imported IL 3 5.3 2.7 64
Domestic DL 2 6.7 3.4 66
Imported IL 4 6.5 4.5 37
a The % difference between the total SO2 for the discrete and segmented flow analyzers is shown. DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid); PRA =
p-rosaniline; SNBC = Sierra Nevada Brewing Company; CL = craft lager; IL = imported lager; and DL = domestic lager.
Methods to Quantify Total Sulfur Dioxide in Beer
Conclusions
For quantifying total SO2 in lager beers for release to the
consumer, DTNB should be utilized over PRA, because even
in a relatively neutral environment (SB), PRA quantified SO2
less accurately than DTNB. The difference in quantified SO2
produced during fermentation was significant, in which PRA
consistently showed lower total SO2 values than DTNB. The
reason behind the difference in quantified SO2 in beer may be
due to adducts other than carbonyls preventing bisulfite from
reacting with PRA.
ACKNOWLEDGMENTS
Thank you to Sierra Nevada Brewing Company Quality Assurance
for their help in this project. Susan Krug performed the initial experi-
ments on lagers and assisted in running the DFA and SFA. Jeff Peter-
son helped run samples on the SFA. Kim Bacigalupo helped with
testing SO2 in other breweries’ lagers.
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