reviewtvbtma_part1
TRANSCRIPT
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EJEAFChe
Electronic Journal of Environmental, Agricultural and Food Chemistry
ISSN: 1579-4377 A CRITICAL REVIEW OF TOTAL VOLATILE BASES AND TRIMETHYLAMINE
AS INDICES OF FRESHNESS OF FISH. Part 1.
Determination.
Peter Howgate
26 Lavender Row, Stedham, Midhurst, West Sussex GU29 0NS, UK
e-mail [email protected]
ABSTRACT. The large literature on the formation of volatile amines, and of their
aggregation as Total Volatile Base (TVB), during spoilage of vertebrate, teleostean fish in
ice, and their used as indices of freshness is critically reviewed. This part reviews analytical
procedures. The thermodynamics of the distillation process in determining TVB is reviewed
and optimum conditions for the distillation are considered. Distillation other than at
temperatures below about 501C is accompanied by decomposition of nitrogen-containing
compounds with the formation of ammonia which additional to the intrinsic ammonia in the
sample. The amount of decomposition differs among the various procedures used for
determining TVB. Rapid distillation in automated distillation units produces the smallest
amount of decomposition. Trimethylamine (TMA) is often determined by the picrate method,
but this is not completely specific for TMA and some dimethylamine is included in the result
depending of the alkali and the amount of formaldehyde used. Other analytical techniques
such as GLC and Flow Injection Analysis are specific and are to be preferred for critical
work. There are differences among procedures for determining TVB and TMA in the way
concentrations in the sample are calculated when using protein-free extracts which leads to
biases between methods.
KEYWORDS: TVB, TMA, DMA, ammonia, distillation, analysis, picrate method
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I. INTRODUCTION
Fish are notorious for spoiling rapidly even when stored under chill conditions. The storage
life to end of good quality for typical demersal, white-fleshed species is around 7 days in ice,
and by about 14 days the fish is unfit for consumption. Equivalent storage lives for pelagic
fish are even less. Measurement of freshness is therefor an important part of quality assurance
of chill-stored fish, and though sensory methods are generally considered to be the most
appropriate for measuring freshness of fish, there is a role for non sensory methods (Botta,
1995; lafsdttir et al., 1997). The chemical test with the longest history of use as an
indicator of freshness is measurement of the amount of basic compounds recovered by
distilling fish muscle, or extracts of fish muscle, under alkaline conditions. The amount of
Total Volatile Bases (TVB) is almost invariable expressed on a nitrogen basis, each
component of the mixture containing one nitrogen atom per molecule, as Total Volatile Base
Nitrogen (TVBN). The former term will be used in this review. The formation of TVB and its
constituent amines in spoiling fish muscle and their relevance as measures of spoilage have
been studied for almost a century now and consequently there is now a considerable literature
on the subject. Though this literature runs to some hundreds of publications including reports
of research, summaries, and discussion papers there are few extensive reviews. Hebard et al.
(1982) wrote a comprehensive review of the subject of methylamines in fish emphasising the
chemistry and microbiology of the formation of amines in chill stored and frozen stored fish
and in fishery products, but also discussing the analysis of amines and TVB and their
suitability as indices of quality. The paper cites 500 or so references. Oehlenschlger (1997)
is a bibliography of methylamines and TVB as measures of spoilage, with a brief review,
which cites 133 references and Botta (1995) has a short discussion of analytical methods for
determination of TVB and its use as a measure of freshness.
The objective of this review is to critically review analytical procedures for measurement of
TVB and amines in fish muscle, their formation in fish, predominately vertebrate fish, during
iced storage, and the effectiveness of TVB and individual methylamines as measures of
spoilage. Part 1 will provide a general introduction to the topic and a detailed review of
methods for their determination, Part 2 a review of formation of volatile bases in spoiling
fishery products and applications in quality assurance.
II. AN HISTORICAL PERSPECTIVE
It seems to have been known in the mid 19th century at least that amines are present in
fishery products. Winkles (1855) isolated amines from spent pickled herring liquor by a
rather heroic preparation starting with 26 gallons, (120 litres), of liquor from which the
amines were recovered by a series of distillations under alkaline conditions with the most
volatile amine being identified as trimethylamine (TMA). (Readers of the paper will note that
the empirical formula of the methyl group is given as C2H3 because at that time the atomic
weight of carbon was considered to be 6; it was revised to 12 in Cannizzaro's revision of
atomic weights in 1860). Winkles' (1855) isolation of TMA was part of a study on amines as
chemical entities and not particularly of amines as components of fishery products, and
systematic investigations of the spoilage of fish and the formation of TVB and amines during
spoilage began in the following century.
Anderson (1908) described changes in sensory properties of salmon during spoilage in ice
and made some microbiological observations, but he did not carry out any chemical analyses.
The earliest paper that the reviewer can find on TVB in the context of spoiling fish is
Tillmans and Mildner (1916) who measured TVB by distillation of fish muscle in the
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presence of magnesium oxide. At about the same time, Clark and Almy (1917) considered
some chemical analyses that might possibly be used to monitor spoilage of fish and tested
some candidate procedures for interference by constituents of fish muscle. The authors
measured TVB by an aeration procedure and obtained complete recoveries of ammonia and
amines from test mixtures, but did not apply this or any of the other tests to samples of fish.
They recommended 'That a study be made of the value of volatile distillation products like
amines, indol, etc. in detecting incipient decomposition in fish.', but did not cite Tillmans and
Midler (1916). Weber and Wilson (1919) used the aeration procedure to determine TVB in
canned sardines and measured the ammonia and methylamines contents in the distillates. A
table in the paper shows the quantities of 'Volatile Alkaline Material' in canned sardines, and,
for some samples, concentrations of ammonia, mono-, di- and tri- methylamines as well, all
on a nitrogen basis. This study was concerned with changes in composition of the canned
products during subsequent storage rather than with spoilage in the raw material, but is
interesting for being the earliest published report of concentrations of methylamines in a fish
product. Tillmans and Otto (1924) examined some chemical tests, including determination of
TVB, for their potential to indicate the onset of spoilage. The text of the paper reports that the
authors compared various distilling procedures with and without addition of baryta and
selected vacuum distillation without baryta as the most stable. This is the only report
encountered by the reviewer of distillation without the addition of an alkalising agent. The
authors do not refer to the procedure as measuring TVB, but as measuring ammonia,
('ammoniak'), concentration. On the basis of the storage of some species of demersal fish at
12-151C the authors concluded that an 'ammonia' concentration of 30 mg (100g)-1
indicated
the beginning of spoilage.
The 1930's was a fruitful decade for studies of volatile amines in fish. In 1936 two papers
were published that are frequently cited in the literature of TVB in fish. Lcke and Geidel
(1935) measured TVB by two procedures: distillation of an aqueous extract of fish muscle
using magnesium oxide as the alkalising agent, and distillation of a sample of minced muscle
suspended in water with magnesium oxide. On the basis of storage of several species of fish
in ice and analysed for TVB by the latter procedure the authors considered the fish no longer
completely unobjectionable, ('einwandfrei'), when the TVB content exceeded 30 mg (100g)-1
.
The latter TVB procedure, distillation of muscle tissue directly with magnesium oxide, or
slight variants of it, has been used to determine TVB in many studies of spoilage of fish and
is often referred to as the Lcke and Geidel (L&G) method, (with variations of the spelling).
Boury and Schvinte (1935) briefly reviewed various sensory, physical and chemical
procedures for measuring freshness of fish, but concentrated on a more detailed study of the
measurement of TVB. The authors remark that it was well known that heating muscle tissue
with alkalis cause formation of ammonia, notably from amides, and they examined various
combinations of distillation conditions and alkalis for the extent of ammonia production
during distillation. They concluded that distillation at 37-381C under reduced pressure with
lithium carbonate as the alkalising agent gave the lowest amount of decomposition ammonia,
and that distillation at normal pressure with magnesium oxide gave TVB values close to
lithium carbonate at reduced pressure. Beatty and Gibbons (1937) briefly discussed chemical
methods for measuring the spoilage of fish and presented data on TVB contents measured by
distillation of muscle tissue with sodium borate under reduced pressure. However, that paper
is more significant for studies on TVB because of it introduced the Conway distillation cell
for measurement of TVB and TMA. Prior to Beatty and Gibbons' (1937) method for
measuring TMA, individual amines in the TVB were measured by cumbersome procedures
involving direct measurement of ammonia content and selective decomposition of the
methylamines by nitrous acid (Boury and Schvinte 1935; Reay, 1937). Reay (1937) also
describes a colorimetric method for determination of dimethylamine (DMA) in fish muscle
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extracts as its copper dithiocarbamate complex. Poller and Linneweh (1926) had cited
publications on the presence of trimethylamine oxide in dogfish and in cephalopods, and also
isolated it from herring muscle, but the significance of their paper for the history of TVB and
volatile amines in fish is in their reporting of studies in another laboratory in Germany, (not
referenced in the paper), showing that spoilage bacteria reduced TMAO to TMA. Further
studies in the 1930's on TMAO content of fish and its reduction to TMA by bacteria during
spoilage confirmed these observations (Beatty, 1937, 1938, 1939; Brocklesby and Riddell,
1937; Cook, 1931; Tarr, 1939; Watson, 1939)1 .
1The title of the paper by Cook (1931) is misleading in that the paper is about TMAO,
not TMA
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By the end of the 1930's the main features of the measurement of volatile amines in fish and
their formation during spoilage had been established: the amount of TVB determined is
affected by the analytical procedure because of decomposition of nitrogen-containing
substances during the distillation step in addition to the volatile amines present, the more
severe the distillation conditions in terms of strength of alkali and temperature of distillation,
the greater the degree of decomposition; the increase in TVB during spoilage is almost
entirely accounted for by the increase of TMA until the fish is very spoiled, (at least in the
species studies up to that time); the TMA is formed by reduction of TMAO by some species
of the bacterial flora of spoiling fish; TMAO appears to be present in the flesh of all species
of marine fish, but not present consistently in flesh of freshwater fish; TMA is not formed
during storage of sterile muscle; TMA content of spoiling fish muscle increases
approximately exponentially with storage time and is highly correlated with overall bacterial
count; the TVB content of typical marine demersal fish at the limit of acceptability as a result
of spoilage is around 30 mg nitrogen (100g)-1
of flesh. In the following decade a colorimetric
method for determination of TMA as its picrate salt (Dyer, 1945; Dyer and Mounsey, 1945)
was developed which considerably simplified the analysis of this amine in fish muscle and
made a significant contribution to subsequent studies on TMA in stored fish and fishery
products and its use as measures of spoilage. There is now a very large literature on volatile
amines in spoiling fish which has contributed to our knowledge of the effect of species and
storage conditions on production of these amines in fish and fishery products though not
significantly adding to or modifying the main features established during the 1930's and listd
above. There have been developments in analytical procedures since the early days of studies
in volatile amines of which the application of gas liquid chromatography (GLC) has been the
most significant, but judging from the literature, and the reviewer's own experience, the
analytical procedures for TVB and TMA most commonly used nowadays in research and
industry have been, and still are, based on those developed in the 1930's and 40's.
III. ANALYTICAL PROCEDURES
A. TVB
1. General Principles
TVB content of fish muscle is an attribute of the composition of the muscle for which the
analytical methodology is implied in its name, not by the components. By definition it must
be determined by a procedure which volatilises the bases which are recovered and
quantitatively determined. The procedure determines the sum of the bases without
discrimination among them, but analysis of the components of the TVB of spoiling muscle
show it to contain predominately ammonia and TMA with, in some instances, a small
contribution of DMA; monomethylamine (MMA) and higher amines are not present, or if so,
only in trace amounts. The component volatile bases could be determined separately and
summed to give an estimate the TVB, but this nullifies the simplicity of measurement of
TVB with regard to both concept and analytical methodology. The difficulty with analytical
procedures for measuring TVB is that fish muscle and extracts of fish muscle contain
compounds that decompose under some distillation conditions to produce ammonia which is
estimated along with the ammonia and amines already present in the sample. The effect has
been recognised from the earliest days of measurement of TVB in fish muscle, and has been
noted in measurement of ammonia by volatilisation in other biological materials (Nichols and
Foote, 1931). Boury and Schvinte (1935) in their extensive study of measurement of TVB
remark that it is well known, ('bien connu' in the original), that ammonia, (notably from
amides), is formed by hydrolysis during distillation. The completeness of distillation of the
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volatile bases increases with alkalinity of the solution or muscle suspension being distilled
and with the temperature of distillation, but these are also the conditions that increase the
extent of decomposition of nitrogen-containing compounds, and practical analytical
procedures attempt to reconcile these competing considerations. This has led to a variety of
procedures being used for determination of TVB, but most fall into one of three major
classes: distillation of a suspension of muscle tissue in water with a moderately mild alkali
such as magnesium oxide (MgO), often referred to as the L&G method when MgO is used;
steam distillation of a protein-free extract of muscle tissue with a strong alkali such as sodium
hydroxide; and diffusion of the bases at room temperature or a little above in a Conway cell.
One factor that affects the rate of distillation of the bases from a distilling mix is the
proportion of the base that is in the unionised form at the pH and temperature of the mix. The
pKas of ammonia, DMA and TMA at 251C are given reference books as 9.25, 10.73, and
9.81 respectively. pKs are temperature dependent and Emerson et al. (1975) derived an
expression for the effect of temperature on the pKa of ammonia in the range 0-501C based on
published information:
pKa = 0.09018 + 2729.2 T-1
, (1)
T in kelvins. The reviewer could not find a corresponding equation for ammonia above 501C,
nor for DMA and TMA at any temperature range, but extrapolating this equation to1001C for
all of the bases gives pKas of 7.41, 8.89, and 7.97 for ammonia, DMA and TMA respectively.
The pH of a suspension of MgO and fish muscle does not seem to have been recorded, but an
estimate can be made from its solubility in water. A search of the Internet produced several
values of the solubility product of magnesium hydroxide, but the most frequently listed value
was 5.6x10-11
at 181, equating to a solubility of 6.7x10-4
M, 0.04 g l-1
. Assuming all of the
dissolved magnesium hydroxide is ionised the pH of a saturated solution of MgO should be
about 10.7 at room temperature. The solubility of the MgO will be greater at the temperature
of distillation, which will raise the pH, but the fish muscle will tend to buffer the mixture and
lower the pH so it is not easy to anticipate the pH of the mix during distillation. Assuming a
pH 10.5 the proportion of the bases in the unionized form are 0.98 in the case of DMA and
more than 0.99 in the cases of ammonia and TMA. Though it would be unwise to take these
extrapolations as accurate predictions of the dissociation of these bases at 1001C they show
the at least the direction and possible size of the effect, and it would appear that incomplete
association of the bases into the free form will not be a factor in the measurement of TVB by
simple distillation using MgO or stronger alkalis as the alkalising agent. Steam distillation
procedures usually specify use of strong alkalis and in these case the bases will be almost
completely in the unionised form. Weaker alkalis giving lower pHs in the distilling mix can
be used, but at the expense of longer distilling times to effect complete recovery (Nichols and
Foote, 1931). The Conway procedure is carried out at room temperature or a little above, but
again strong alkalis are used and the proportion of TVB bases in the unionized form will be
greater than 0.99.
Another factor that affects the volume of distillate required for complete recovery of a base,
and hence the distillation time, is its volatility, a property measured by the Henry's law
constant for the base. Egnr and Johansson (1938) made a theoretical study of the distillation
of ammonia as performed in the final step in the Kjeldahl determination of nitrogen, and for
simple distillation of volatile substances from an aqueous solution they derived an expression
for relating the proportion of a volatile substance distilled over to the proportion of the
original distillation solution distilled over. Rearranging the equation as given in the original
paper, and using different symbols, a form of their equation is:
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(1-Pm) = (1-Pv)Q (2)
where Pm is the proportion of the original mass of volatile substance present in the distillate
and Pv is the proportion of the original volume of distilling solution distilled over. Q is a
volatility coefficient equal to H/k where H is the dimensionless Henry's law constant, the
ratio of concentration of the substance in the vapour phase to that in the liquid phase, and k is
the ratio of the specific volume of water to that of saturated steam at the temperature of
distillation. Values of k can be obtained from standard steam tables and at 1001C is 6.24x10-
4. Egnr and Johansson (1938) estimated a value of 80x10
-4 for H for ammonia at 1001C
from published data giving a value of Q of 12.8. They studied the distillation of ammonium
solutions in water using MgO as the alkaliser and obtained an average value for Q of 14.4,
equating to a value of 90x10-4
for H, in reasonable agreement with the assumed value. Sander
(1999) has compiled data on the Henry's law constants for very many chemicals and lists 15
values of the constant for ammonia. According to information in the tabulation some of these
values are secondary data, that is, appear in reviews or similar, and some have not been
checked by the compiler. Four values are shown as measured values and their mean is
6.04x10-4
at 251C when converted to the dimensionless constant from the units used in the
compilation. The compilation reports that tabulated values, which are given for 251C, can be
converted to values at other temperatures using the van't Hoff relationship and tabulates
appropriate coefficients for the equation. Using the mean of three measured values of the
coefficient for ammonia the Henry's law constant for ammonia at 1001C is calculated to be
83.7x10-4
, equating to a value of 13.4 for Q compared with the observed value of 14.4. (There
are reasons, discussed below, to expect the value of Q, and hence H, observed in simple
distillation experiments to be greater than the true value depending on the conditions of
distillation). Using the expected value of Q, 13.4, in equation (2) it can be calculated that
99% of the ammonia in a solution will be distilled over when the distillate volume is 29% of
the volume of liquid originally in the distilling flask.
Sander (2007) lists single, measured, values of H for TMA and DMA of 42.6x10-4
and
7.47x10-4
at 251C respectively when converted to the dimensionless constant. The value of H
for TMA is very much greater than the corresponding value for ammonia and all of the TMA
would be recovered in the volume of distillate required for recovery of ammonia. The values
of H for ammonia and DMA are very similar at 251C and whether or not the volume of
distillate required for recovery of ammonia would suffice for recovery of DMA will depend
on the relative sizes of the temperature coefficient in the van't Hoff equation, but
unfortunately, measured values are not included in the Sander (2007) compilation.
2. Direct Distillation of Fish Muscle
In the procedure described in Lcke and Geidel (1935), the usual citation for the
determination of TVB by direct, (as distinct from steam), distillation of fish muscle, 5-10g of
sample is suspended in 300ml of water and distilled with MgO. Using the calculations above
this starting volume would require a distillate volume of 87ml for recovery of substantially
all, 99%, of the ammonia. The original paper specifies the time of distillation - 25 minutes
following 10 minutes to reach boiling - but not the volume of distillate or the distillation rate.
The only publication the reviewer has come across relating to distillation of individual
methylamines in the context of TVB determination by any procedure is in Hjorth-Hansen and
Bakken's (1947) detailed review of analytical procedures for measuring amines in fish and
fish meal. They measured the time course of the distillation of ammonia and methylamines
from aqueous solutions using MgO as the alkali. Figure 3 of the publication shows a plot of
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the proportion of base distilled over against the distillation time, starting from when the
distilling liquid started to boil, as smooth curves without experimental points, but the text
does not report the volumes of the distillates, nor the distillation rate from which the volumes
can be calculated. The authors cite the Egnr and Johansson (1938) paper and Figure 4 of
Hjorth-Hansen and Bakken (1947) shows a plot of the fraction of ammonia distilled over
against the volume fraction of distillate in the range 99.5% to 99.9% of ammonia distilled
over computed from the Egnr and Johansson equation using the value of 14.4 for Q from
that paper, but it is not possible to infer from this curve the distillation rate used in
determining the experimental curve for ammonia. (The Egnr and Johansson equation
presented in Hjorth-Hansen and Bakken (1947) has a typographic error in that the
(division) symbol in the quoted formula should be a (minus) symbol). In the case of
ammonia and using equation (2) with a value of 13.4 for Q the expected value of Pv for Pm =
0.95 is 0.20. The original starting volume used in Hjorth-Hansen and Bakken (1947) was
250ml so the volume of distillate at this point would be 50ml. The ammonia distillation curve
in Figure 3 shows a Pm of 0.95 being obtained at a distillation time of 20.5 minutes giving a
mean distillation rate to that point of around 2.5 ml minute-1
. The distillation curves in
Hjorth-Hansen and Bakken (1947) show TMA distilling over much faster than ammonia as
would be expected from relative values of H at 251C. 95% of the TMA is shown as distilling
over at 9.1 minutes and using the assumed distillation rate of 2.5 ml min-1
a value of Q of
31.3 for this base is predicted. Though their values of H are similar at 251C the data shows
DMA distilling over much slower than ammonia does and 95% of the DMA is recovered in
43.6 minutes equating to a value of 5.23 for Q assuming a distillation rate of 2.5 ml min-1
.
From these measured and estimated values of Q it can be calculated that the fraction of the
original distilling solution to be distilled over, Pv, for 99% recovery of TMA, ammonia and
DMA are 0.14, 0.29 and 0.59 respectively. If the sample is suspended in 300ml of water, the
conditions of the Lcke and Geidel (1935) procedure, the corresponding volumes to be
distilled over are 41ml, 87ml and 176ml. These values should be considered maximum
estimates and in practice the actual volumes required to be distilled will be less than these.
Egnr and Johansson (1938) pointed to some aspects of experimental procedures that would
reduce the amount of distillate required to recover a given proportion of the compound being
distilled, the most important of which for the case of the L&G procedure would be refluxing
in the distillation apparatus. The effect of refluxing is to increase the concentration of base in
the vapours reaching the receiving flask compared with the situation without any refluxing
and Egnr and Johansson (1938) derived an equation relating the degree of enrichment to the
proportion of water returning to the distillation flask due to refluxing. This enrichment results
in an effective increase in the value of Q and Egnr and Johansson (1938) demonstrate the
effect with experimental studies using different distillation assemblies. This reflux effect is
apparent in the Hjorth-Hansen and Bakken (1947) data when the curve for ammonia in their
Figure 3 is compared with the curve calculated from equation (1) using a distillation rate of
2.5 ml min-1
. At the start of the distillation the proportion of ammonia distilled over is greater
than predicted from the equation and the deviation decreases as distillation proceeds.
Hjorth-Hansen and Bakken (1947) recorded their distillation times from when the distilling
solution started to boil, a requirement of the L&G procedure, and refluxing would be greater
at the start than later when the splash head and distillation head have warmed up. There is
insufficient information in the Hjorth-Hansen and Bakken (1947) paper to calculate the effect
of refluxing, but judging from the examples in Egnr and Johansson (1938) the consequence
is probably to reduce the volume required for 99% recovery of the bases to by at least 10% of
the volumes quoted above.
It follows from theoretical considerations of distillation discussed above that protocols for
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determination of of TVB by simple direct distillation should specify the volume of distillate
rather than time of distillation. The procedure specified in Lcke and Geidel (1935) requires
that the distilling liquid be brought to boiling in 10 minutes and if using their apparatus this
would specify the distillation rate and hence the volume of distillate recovered in the 25
minutes distillation time. However, deviations from these standard conditions could lead to
errors in the TVB measurements. It would appear from the discussion above that recovering
29% of the original in the distillate will recover more than 99% of the TMA and ammonia,
but only about 85% of the DMA. However DMA is a minor constituent in TVB and less than
complete recovery of this base would not appreciably affect the accuracy of TVB
measurement. It follows from the theoretical treatment of distillation that a smaller volume of
original distilling liquid will require a smaller volume of distillate to recover 29% in the
distillate. Using an original volume of 100ml, for example, rather than the specified 300ml
will require only 9 minutes distillation time at most rather than 25. Apart from any saving in
time the reduced reaction time will result in a smaller amount of ammonia being formed by
decomposition, (see discussion below), thus leading to a measured TVB value closer to the
true value.
An alternative procedure to direct distillation is steam distillation. The semi-micro distillation
glassware commonly using in the Kjeldahl procedure for nitrogen determination is not
suitable because it does not cope well with solid material, and usually more basic glassware is
needed. Antonacopoulos (1960) has described an apparatus for steam distillation of
foodstuffs which has been extensively used for determination TVB in fish and is often
referred just as the 'Antona apparatus'. The glassware is not typically a stock item in
catalogues of laboratory suppliers and usually has to be made to order. The sample of minced
fish is transferred to a distilling tube with a small amount of water, MgO added, the tube
inserted into a flask of water acting as a steam generator, and steam distilled
(Antonacopoulos, 1960, 1968; Antonacopoulos, 1973; Antonacopoulos and Vyncke, 1989).
The procedure specifies distillation at 10 ml min-1
. for 12 minutes giving a distillation volume
of 120ml. The traditional glassware used in the distillation step of the Kjeldahl determination
of nitrogen is giving way to semi-automatic, rapid, distillation units and this equipment can
be used for distillation of muscle samples with MgO (Malle and Tao, 1987;Vyncke, 1996;
Surti et al., 2001; European Commission, 2005) and is now a convenient alternative to the
Antona apparatus. During steam distillation the distilling mix does not decrease in volume as
it does in simple distillation and Egnr and Johansson (1938) developed another expression
for this case:
(1-Pm) = e-QPv
(3)
Symbols the same as equation (2). Inserting the value of 13.4 for Q in the equation predicts
that the volume of the distillate for 99% recovery of ammonia and TMA needs to be 34% of
the volume of the distilling solution. In the case of steam distillation for determination of
TVB the distilling volume is typically in the range 25-50ml leading to sufficient volumes of
distillate in the range 9 -18ml. All the TMA will be recovered in this proportion, but the
volumes needed for recovery of DMA will be about two and half times these, 22.5 - 45ml.
The 120ml specified when using the Antona apparatus then seems excessive even to ensure
recovery of DMA.
3. Distillation of Fish Muscle Extracts
Distillation of solid samples has some drawbacks in practice in that the distillation apparatus
has to able to handle solid samples, and the apparatus has to be dismantled at the end of the
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distillation to remove the solids. These considerations often lead to bulky and unwieldy
apparatus and a slow throughput of samples though the Antona apparatus and rapid
distillation units overcome these. Especially the rapid distillation units because these can
complete the distillation in 5-10 minutes and tubes can be prepared while other samples are
being distilled. An alternative is to prepare extracts of the fish muscle and use semi-micro
Kjeldahl glassware for the distillation, invariably by steam distillation. Distillation times are
in the order of 10 minutes and throughput is rapid especially if glassware that automatically
siphons off the distillation mix at the end is used. Use of extracts does require extra
processing steps, but there are advantages in that large sample weights can be used so
reducing sample variance, and the extracts might be required for another analyses anyway.
Another benefit is that much of the nitrogen-compounds with the potential to decompose to
form ammonia are removed from the distillation matrix.
Some of the earlier studies on TVB investigated use of aqueous extracts (Tillmans and Otto,
1924; Hess, 1932; Boury and Schvinte, 1935; Lcke and Geidel, 1935; Hillig et al., 1958) or
press juice, the liquid squeezed from muscle by pressure, (Beatty, 1938; Watson, 1939), but
more often protein-free extracts of various sorts have been used. The use of acidic colloidal
ferric hydroxide has been described by Tillmans and Otto (1924), Hjorth-Hansen (1952) and
Hjorth-Hansen and Bakken (1947), magnesium sulphate by Tomiyama et al. (1956),
formaldehyde by Dyer and Mounsey (1945), and ethanol by Stansby et al. (1944), but use of
these precipitants have rarely been reported outside of the original papers. By far the most
frequently used protein precipitants used in determination of TVB and volatile amines in fish
are trichloroacetic acid (TCA) and perchloric acid (PCA). TCA seems to have first been used
in analysis of TMA fish muscle in Canadian laboratories (Dyer and Mounsey, 1945) and
rapidly taken up in other laboratories. The concentrations used are usually 5 or 7.5% at a ratio
of usually 1:2 or 1:3 fish muscle to extractant. PCA was first used in studies on the post
mortem biochemistry of fish muscle (Saito and Arai, 1958; Jones, 1960), (the perchlorate ion
can be removed as its insoluble potassium salt, whereas TCA needs to be removed by an
organic solvent), though the extracts can be used for determination of TVB and amines. The
concentration used is almost invariably 6%, (0.6M), and ratios 1:2 to 1:9 fish:extractant have
been used in making the extracts. An extract prepared by grinding muscle with solid TCA can
also be used (Shewan et al., 1971). Sodium hydroxide is the common alkalising agent, but
MgO (Vyncke et al., 1987) and calcium hydroxide (Hillig et al., 1958) have been used.
An aspect of the use of extracts for determination of TVB on which there is lack of
uniformity is that of calculating the TVB concentration in the muscle tissue. (And when
calculating individual bases in extracts for that matter). Muscle tissue is blended with the
extractant, the TVB determined in an aliquot of the extract, and the concentration in the
aliquot scaled up to give the concentration in the tissue. In some descriptions of procedures
the scaling factor is the ratio of the total mass of sample plus volume of extractant to the
volume of aliquot, but, on the basis that the bases are dissolved in the aqueous phase only, the
factor should be the ratio of the sum of the water in the sample of fish tissue and the volume
of extractant to the volume of aliquot. Using total mass rather than volume of aqueous phase
in the scaling factor results in an overestimate of the TVB concentration, (or the
concentration of other bases if their analyses are based on extracts), by an amount which
depends on the ratio of muscle sample to extractant volume and the water content of the
sample. For example a common ratio for extraction with TCA is 1 part fish:2 parts of TCA
solution; if total mass is used the scaling factor it is 300/100, but is 280/100 if water masses
are used and the water content is 80%, an overestimation by a factor of 1.07 when the total
masses are used. Boury and Schvinte (1935) pointed out that the scaling factor should be
based on the volume of water phase rather than total mass, but the advice has not always been
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11
followed by other workers. In fact many of the descriptions of procedures used for
determination of TVB do not include an explicit statement about calculation of concentration
in the fish tissue, and even official or semi-official recommended procedures differ. Some
papers on TVB in fish use what the authors refer to as the "Codex" method and cite a
document presented to the Codex Committee on Fish and Fishery Products at its 3rd session
in 1968 and referenced as Codex Fish 1/7. This document prescribes a scaling factor based on
total mass. It is difficult to obtain a copy of this document because the report of the meeting
is not readily available, but the experimental procedure is described in detail in Vyncke et al.
(1987) B steam distillation of a TCA extract with sodium hydroxide. The description though
does not give the formula for calculating the TVB concentration, but presumably the
recommendation in the Codex document applies. The TVB procedure in the recommended
methods for the analysis of fish products of the Analytical Methods Committee (1979) uses a
scaling factor based on the volume of the water phase and this procedure is included, in
summary, in Egan et al. (1981). The Official Methods of Analysis (Association of Official
Analytical Chemists, 1995) does not have a method for TVB, but the recommended method
for TMA determination, method 971.14, utilises a TCA extract as is used in TVB
determinations, and the calculation for TMA concentration in the original sample uses a
scaling factor based on total mass. FAO (1998) uses a scaling factor based on water volumes
in the procedure for TVB in and cites Egan et al. (1981), but in the procedure for
determination of TMA uses the mass ratio for calculating TMA citing the AOAC procedure
(Association of Official Analytical Chemists, 1995). The procedure for TVB determination
recommended in EU legislation (European Commission, 2005) uses distillation from a PCA
extract with the scaling factor being based on total mass, though in this case the
overestimation is negligible as the extract is made at a 1:9 fish:extractant ratio.
4. Distillation under reduced pressure
Decomposition of nitrogen-containing substances during direct or steam distillation can be
avoided, or at least reduced substantially, by carrying out the distillation at low temperature B
compared with 1001C B under reduced pressure (Tillmans and Otto, 1924; Boury and
Schvinte, 1935; Tomiyama and Harada, 1952; Tomiyama et al., 1956; da Costa et al., 1960;
Hjorth-Hansen and Bakken; 1947; Pearson and Muslemuddin, 1968). The distilling bath is
immersed in a water bath at perhaps 50 or 701C, but because of evaporative cooling the
distilling solution will be below that of the water bath. Both muscle extracts and suspensions
are amenable to distillation under reduced pressure and recovery of TVB is quite rapid; for
example Pearson and Musselmuddin (1968) specify distillation of muscle suspension with
MgO for 25 minutes at a pressure of 10-15mm of mercury and a bath temperature of 501 and
Tomiyama et al. (1956) using a water bath at 701C reported that most of the TVB distilled
over in 5 minutes. The disadvantages of distillation under reduced pressure rather than at
atmospheric pressure are the more complex apparatus required and the longer time taken to
dismantle and reassemble the apparatus between analyses. These disadvantages seem to deter
potential users because distillation at reduced pressure does not appear to have been used
outside of the laboratories describing the procedures.
5. The Microdiffusion (Conway) Method
What is almost invariably referred to as the 'Conway' method is a popular procedure for
measuring TVB judging from published reports; a survey, not comprehensive, by the
reviewer more than 100 papers on TVB in fishery products revealed that almost half had used
the method in the studies. Distillation takes place at room temperature or a little above in a
circular microdiffusion cell B the Conway cell B which has a central well bounded by a wall
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12
a little lower than the outer rim of the cells and forming an annular well. The sample, an
extract of fish, is mixed with alkali in the annular well and acid is placed in the central well to
absorb the evolved bases. The Conway cell seems to have been first used in studies on
spoiling fish muscle by Beatty and Gibbons (1937) to measure TMA, but the procedure was
soon adopted to measure TVB. The first explicit account of the use of the microdiffusion
technique for TVB seems to be Stansby et al. (1944) who did not employ the Conway cell as
such, but a similar cell assembled from other glassware. The Conway method probably
enjoys its popularity because of the simplicity of the experimental procedure and the low cost
of the equipment. The dwell time for complete absorption is two hours at room temperature
when saturated potassium carbonate is used as the alkaliser or one hour at 371C, but these
times are acceptable in practice. Montgomery (1960) described a rapid procedure based on
the time taken to neutralise a fixed amount of acid.
The drawbacks of the procedure reside in the care necessary in its execution for accurate and
precise results. Because only small amounts of base are involved the analyst must be
experienced in titrating from a microburette, for example in applying a thin layer of stopcock
grease or similar to the tip of the burette to reduce the drop size and near the end point wiping
less than a drop from the tip with the stirring rod. The glassware must be scrupulously clean
even to the extent of cleaning the Conway cells in chromic acid between analyses, and the
sealing grease must be carefully applied, or flamed, to exclude air bubbles. The standard
sodium hydroxide for back titrating the absorbing acid B if standard mineral acid rather that
boric acid is used B must be carbonate-free. General texts on the procedure, for example,
Conway (1950) describe the principles and theory of absorption in the Conway cell and the
practical requirements for accurate and precise analysis. Spinelli (1964) has discussed some
of the precautions required for accurate results in the context of using the Conway method for
measurement of TMA, but they apply equally to the determination of TVB.
6. Decomposition During Distillation
It seems to have been known from early days of measurement of TVB in fish that ammonia is
formed during distillation and is included in the value of TVB obtained in an analysis, and it
was soon established that the amount of decomposition depended on both temperature of
distillation and on the pH of the distilling mix (Boury and Schvinte, 1935; Hjorth-Hansen and
Bakken; 1947). The general conclusions of those two studies for minimisation of
decomposition ammonia were that intact muscle should be distilled under vacuum with MgO
or weaker alkali and that protein-free extracts could be distilled at atmospheric pressure with
MgO, but not with strong alkalis.
Pearson and Musselmuddin (1968, 1969a, b) made a series of detailed studies on the rates of
formation of decomposition ammonia during the determination of TVB in fish by distillation
of muscle suspensions of salmon, plaice and haddock at 501C under reduced pressure and at
atmospheric pressures with MgO as the alkaliser, (the latter being the classic L&G
procedure). They measured the amount of bases distilled from the samples up to 60 minutes
distillation with sampling of the distillate at intervals. The figures in the papers of the time
course of distillation of bases show an initial curvilinear phase corresponding to the
distillation of the intrinsic bases followed by linear phase after about 20 minutes of
distillation corresponding to the continuing formation of ammonia by decomposition. The
authors report that the rates of decomposition are very much less during distillation under
reduced pressure than at atmospheric pressure, though not zero, and do not differ appreciably
among the species. The reviewer has made a further analysis of the data by assuming a
mathematical model for the course of distillation:
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13
TVBt = A(1-expkt
) + bt (3)
where TVBt is the measured TVB concentration at distillation time t, A is the amount of
intrinsic TVB and k and b are rate coefficients. The first term represents the distillation of the
intrinsic TVB to an asymptote, A, and the second to the decomposition ammonia increasing
linearly with time. (The decomposition should also be represented by an asymptotic
expression, but over the time course of the experiment a linear expression is adequate). Data
were read off the figures in Pearson and Musselmuddin (1968, 1969a, b) and the model fitted
to each set, species by storage time by procedure, to estimate values of the coefficients that
minimised the sums of squares of deviations of calculated values from the observed values.
There were insufficient data values to accurately determine the errors of the estimates, and
hence the significance of any differences, but within a procedure there was no suggestion that
species or storage time influenced the rate coefficients, though, as would be expected, the
estimates of A, the asymptote, increased monotonically with storage time. The mean values
of k, the rate coefficient for distillation of intrinsic TVB, over all species and storage times
were 0.25 mins-1
and 0.45 mins-1
for distillation at atmospheric and at reduced pressures
respectively. Distillation by the procedure at reduced pressure is much faster than that at
atmospheric though the procedure as described in Pearson and Musselmuddin (1968)
specifies 25 minutes distillation, the same as that at atmospheric pressure. Using the rate
constants quoted above it can be calculated that 99% of the intrinsic TVB is distilled over in
19 minutes at atmospheric pressure and 10 minutes at reduced pressure. The rate coefficients
for the degradation reaction were 0.34 and 0.054 mg TVB (100g)-1
min-1
for distillation at
atmospheric and reduced pressures respectively. These values are within the ranges quoted in
the Pearson and Musselmuddin (1968, 1969a, b) papers for the rates at atmospheric pressures,
and about the same as the upper value of the range, 0.02-0.05, for distillation at reduced
pressure. Vyncke (1970) reports a similar rate, 0.4 mg TVBN (100g)-1
min-1
, for distillation
of muscle suspension with MgO for between 20 and 60 minutes in the Antona apparatus at
atmospheric pressure, and unpublished data from Torry research Station, Aberdeen, UK,
(Howgate personal experience), gave a value of 0.36 mg TVBN (100g)-1
min-1
. Using the
rates estimated from the Pearson and Musselmuddin (1968, 1969a, b) papers the amount of
decomposition ammonia generated by a 25 minutes distillation time are 8.6 and 1.4 mg
nitrogen at atmospheric and reduced pressures respectively. Distillation time at reduced
pressure under the conditions described in Pearson and Musselmuddin (1968) could be
reduced to 15 minutes with over 99% recovery of the intrinsic TVB with an associated
reduction in the amount of decomposition ammonia nitrogen to 0.8mg. Pearson and
Musselmuddin (1968) reported that raising the temperature of the water bath from 501 to
701C increased the rate of decomposition, but did not provide any data on the rate at the
higher temperature. It would appear that distillation at a bath temperature lower than 501C
would reduce the rate of formation decomposition ammonia even further, but with a
concomitant requirement to increase in the distillation time. On balance there might not be
any practical advantage to do so.
There seems no doubt that amides, glutamine and asparagine in particular, are the major
source of the decomposition ammonia during distillation of whole muscle (Rehbein and
Oehlenschlger, 1988). The mean amide content of the muscle protein fraction of four
species of teleost fish reported by Connell and Howgate (1959) was 6.24mg amine N (100g)-1
protein N. Assuming the total nitrogen content of teleost fish muscle is around 2.8g (100g)-1
of which about 85% is protein nitrogen, the amide nitrogen content is then around 150mg
(100g)-1
muscle tissue, very much more than is needed to account for the observed
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14
decomposition ammonia in the TVB procedure. Muscle tissue of elasmobranch fish contains
large amounts of urea which is readily decomposed to form ammonia by even mild alkalis
and generally TVB measured by distillation at atmospheric pressure is not recommended as a
measure of spoilage for this type of fish. However, Pearson and Musselmuddin (1969b)
showed that distillation under reduced pressure using MgO as the alkaliser does not result in
decomposition of urea and provides a true measure of intrinsic TVB in elasmobranch muscle.
The formation of decomposition ammonia from amides in protein can be avoided by using
protein-free extracts though other nitrogen-containing compounds in such extracts can
decompose under appropriate conditions. The glutamine content of cod protein-free extracts
was reported by Mackie and Ritchie (1974) to be 1.4mg (100g)-1
of muscle tissue, equivalent
to 0.12mg amide nitrogen (100g)-1
, a negligible amount in the context of TVB content.
Pearson and Musselmuddin (1969) has a plot of the time course of TVB content of haddock
TCA extract by the L&G procedure which shows no increase in TVB after the initial 20
minutes. Oehlenschlger (1988) showed that the higher the pH of the alkalised PCA extract,
measured at room temperature, the greater the rate of deamination during distillation. He
found the rate of decomposition when using NaOH as the alkali was 1.3 mg N (100g)-1
(50ml)-1
distillate in the case of redfish (Sebastes marinus) and 3.1mgN (100g)-1
(50ml)-1
distillate in the case of cod (Gadus morhua). The distillation rate in the Antona apparatus is
typically 10 ml min-1
so the decomposition rates are 0.26 and 0.62 mgN (100g)-1
min-1
for
redfish and cod respectively. Rehbein and Oehlenschlger (1982, 1988) reported using the
L&G method, (presumably using MgO as the alkaliser though the paper is not explicit on this
point), with the Antona unit to measure TVB in PCA extracts and also measured the
ammonia and methylamine contents of the extracts. They found there was rapid formation of
decomposition ammonia during the time the intrinsic TVB distilled over and some
continuous formation of decomposition ammonia on further distillation which was almost
negligible in fresh fish but greater in spoiled. Some unpublished results from Torry Research
Station (Howgate, 1993) confirmed these findings of rapid initial formation of decomposition
ammonia followed by slow continuous formation at a rate which was greater in stale than in
fresh fish.
7. Other methods
Measurement of TVB by distillation at room temperature has the advantage that there is no,
or only negligible, formation of ammonia by decomposition of nitrogen-containing
compounds. The distillation is commonly carried out in the Conway cell, but that has some
disadvantages experimentally, as described above, mostly associated with its being a micro
method. Alternatively the distillation can be effected on a macro scale by aeration of the
alkalised mixture as Clark and Almy (1917) suggested in their early consideration of methods
that had potential for assessing spoilage of fish. Egnr and Johansson (1938) has a discussion
of theoretical aspects of aeration and Hjorth-Hansen and Bakken (1947) made further studies
to compare theory and practice. Stansby et al., (1944) included aeration in their comparison
of methods for determination of TVB, but otherwise the method has not been used in studies
of TVB in fish.
TVB can be measured by Flow Injection Analysis (FIA) (Wekell, et al., 1987; Hollingworth
et al., 1990; Ruiz-Capillas and Horner, 1999; Baixas-Nogueras et al., 2001) in which the
bases are partitioned from an alkalised extract through a PTFE membrane into a carrier
stream containing bromothymol blue indicator. The change in colour of the indicator is
monitored and provides a measure of the total bases. An advantage of the procedure is that it
is carried out at room temperature and should measure the intrinsic TVB content. Pivarnik et
al. (1998) tested the ammonia electrode in its standard configuration as a TVB electrode and
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15
obtained a good correspondence between measurements of TVB in fish by electrode and by
distillation. A collaborative trial of the method (Ellis et al., 2000) revealed that some
laboratories experienced practical difficulties with the procedure, and there were moderately
large discrepancies among results from the participating laboratories. There have been
proposals for simple devices for measuring TVB in head spaces by colour changes of
membranes impregnated with a suitable pH-sensitive dye (Hungerford et al., 1997; Loughran
and Diamond, 2000), but they do not appear to have been adopted in practice.
8. Relationship Between Methods
The measured TVB content consists of the intrinsic TVB in the sample plus the
decomposition ammonia formed during distillation. The results of determination of TVB by
two methods on the same samples should then be related by the expression
TVB1 = TVB2 + c (4)
where c is the difference in the amount of decomposition by the two methods. If a matrix of
values of c were determined for pairs of procedures then values determined by one procedure
could be corrected to those expected of another. Such an approach would suppose that c does
not also depend on species of fish or type of fish product as would happen if the components
that decompose to form ammonia in some procedures varied by species or product.
Vyncke (1970) compared 200 paired values for TVB content, (species of fish not stated), by
the L&G and the Codex methods and calculated the regression equation Y = 0.93X + 5.30,
where Y is TVB content by the Codex method and X the corresponding value by the L&G
method. The regression coefficient is appreciably different from the value of 1.0 to be
expected from the expression quoted earlier, but, as has been pointed out above, the Codex
procedure uses a scaling factor based on total mass which overestimates the true
concentration. A 1:2 fish:extract ratio is used in the Codex method which leads to a 7%
overestimate of the TVB concentration assuming an average water content in the samples of
80%, and when the Y values, Codex method, are corrected by this factor the regression
equation becomes Y = 0.99X + 5.67. This regression coefficient is practically not different
from 1.0 and it seems from this data set that the Codex method, and by extension any
procedure based on steam distillation of a protein-free extract, is biased by approximately
+6mgN (100g)-1
compared with the L&G procedure.
Pearson and Muslemuddin (1971) compared results of TVB determination by distillation
under reduced pressure (Pearson and Muslemuddin, 1968) and by the L&G procedure for 15
samples of fish covering three species, cod, haddock and plaice, and expressed the
relationship as the exponential equation Y = 0.223X1.31
where Y is the value by distillation
under reduced pressure and X those the L&G procedure. The authors used this expression to
convert quality class limits based on the L&G procedure to corresponding values using
vacuum distillation. The authors do not report the original data on which the expression was
derived, but earlier papers (Pearson and Muslemuddin, 1968; 1969a) between them tabulate
nine sets of data obtained by the L&G, vacuum distillation, and Conway procedures on the
same samples of three species of fish, salmon, haddock, and plaice. (The determinations by
the Conway method were carried out on a TCA extract and the authors report they corrected
the results for water content of the samples so that they corresponded to those by the direct
distillation procedures). Calculating the regression coefficients by the usual least squares
procedure when both variable variables are subject to observational error, as they are here,
results in a biased, smaller, estimate of the regression coefficient, and instead the reviewer
calculated the structural relationship between the three pairs of relationships (Kendall and
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16
Stuart, 1967). In all cases the slopes of the structural relationships were not significantly
different from 1.0 and in the case of Conway and distillation under reduced pressure the
constant, c in the expression above, was not significantly different from 0.0. The constants for
the relationships of L&G against either the Conway or the distillation under educed pressure
procedures were significantly different from 0.0 and the mean bias of L&G compared with
the other two procedures was +11.9mgN (100g)-1
of sample. The reviewer also fitted the data
of the L&G and the distillation under reduced pressure methods to Pearson and
Musselmuddin's (1971) exponential model, again as a structural relationship. The exponential
model resulted in a slightly lower residual variance compared with the linear model, but
when tested as an F ratio the difference was not significantly different (p=1.9) and the
simpler linear model can adequately represent the relationship between the methods.
Stansby et al. (1944) used 60% ethanol to prepare protein-free extracts and measured TVB in
these extracts by the Conway method and by direct distillation using sodium borate as the
alkaliser. They compared TVB contents of nine samples of silver salmon stored in ice or at
room temperature by these two procedures and also on press juice from the same samples
using the Conway procedure. The data are shown in Table 1 of the paper and were used by
the reviewer to calculate the structural relationship between pairs of procedures. Results for
press juice by the Conway method were expressed on a 100ml of press juice basis in the
paper and were corrected to a 100g basis assuming the fish contained 80% water. For all
three comparisons the structural relationships were statistically significantly different from
1.0 suggesting systematic differences in what was being measured by the three procedures
and not just biases arising from decomposition. Davidovich and Giannini (1984) measured
TVB in Patagonian hake (Merluccius hubbsi) stored for up to nine days in ice by the L&G
and Conway methods and obtained the relationship Y = 2.37X + 5.27 where Y is TVB
content by the L&G method and X the corresponding value by the Conway method. The
regression coefficient is much greater than is to be expected from the simple linear
relationship above, and judging from the scatter diagram of values by the two methods
included in the paper the error bounds can not include a value of 1.0 even if the structural
relationship were calculated. It is possible there is a systematic error operating here and it is
worth noting that the higher values of TVB by the L&G method shown in a figure in the
paper, around 33mgN (100g)-1
, are about twice those reported by Lupin et al. (1980) for the
same species stored in ice for nine days. Malle et al. (1989) compared TVB values
determined by rapid distillation of a TCA extract with NaOH and by the Conway method for
261 samples and obtained a regression of Y = 1.017X + 0.85 where Y is TVB content by the
rapid distillation procedure and X the corresponding value by the Conway method. Both
procedures utilised TCA extracts and the authors used scaling factors based on the mass ratio
rather than volume ratio. This would not have affected the value of the regression coefficient
if the same extraction ratio had been the same in both, but they used a ratio of 1:2 for the
rapid distillation method and 1:1 for the Conway method. Assuming on average the samples
contained 80% water, (the authors do not give information about the species used other than
they were marine fish), and correcting by the scaling factors based on volume ratios for the
two procedures gives a regression coefficient of 0.982. This is very close to the expected
value of 1.0, especially bearing in mind that the least squares regression procedure provides a
biased, and lower, value of the coefficient compared to that of the structural relationship.
Botta et al. (1984) compared six methods for determining TVB by measuring the TVB
contents of the same samples of ice-stored cod during storage. The procedures were direct
distillation of muscle with MgO the essentially the L&G procedure, steam distillation of
muscle with MgO in a rapid distillation unit, the Conway procedure on a TCA extract,
distillation of a TCA extract with NaOH in a semi-micro distillation unit, distillation of an
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17
acidic magnesium sulphate extract with NaOH under reduced pressure (Tomiyama et al.,
1956), and distillation of an ethanolic extract with sodium borate as described by Stansby et
al. (1944). The test material was cod stored in ice for up to 18 days and the TVB contents
were measured on the same sample by each of the six procedures. The results are shown in
Figure 2 of the paper and shows the TVB contents increasing exponentially with storage time
following a dwell without any increase. (Changes in TVB during spoilage will be discussed
in more detail in Part 2 of this review). This behaviour can be modelled by an expression of
the form:
Y = a + ek(t-d)
(5)
where Y is the TVB concentration, a is the initial value of TVB at start of storage, k is the
rate constant, t is the storage time, and d is the dwell before the exponential growth phase of
TVB. The TVB contents were measured by the different procedures on the same sample of
fish so if the value obtained consists of the intrinsic TVB content of the sample plus the
ammonia formed by decomposition then values of the parameters k and d should be the same,
within experimental error, but values of a will differ among procedures. Figure 2 of
Botta et al. (1984) shows a scatter diagram of TVB contents against storage time, in days and
the values were picked off the diagram and the model fitted to the data by minimising the
sum of squares of deviations of the observed from the fitted values. Inspection of the curves
in the original figure and consideration of the values of the parameters obtained from fitting
the model suggested the results for the Stansby et al. (1944) procedure, distillation of an
ethanolic extract, were anomalous compared with the others. The fitted value of a, the initial
value, was 2.0mgN (100g)-1
compared with 7.4mgN (100g)-1
by the Conway procedure, the
method that is considered not to produce any ammonia by decomposition, and the rate
constant was appreciably lower than those of the other methods. Botta et al. (1984) record
that recovery of a mixture of TMA and ammonia added to the fish sample was only 72% in
the case of the Stansby et al. (1944) procedure. The results for this set of were therefor not
included in further processing of the data. The estimated values of d and k for the other
methods were very similar and a reduced model using the same values of these two
parameters was fitted to all the data giving an estimate of d of 5.0 days and of k of 0.28
days-1
. The combined residual mean square of this model was larger than that of the full
model, but the difference, tested as an Analysis of Variance, was not significant. However a
reduced model in which all three parameters were the same gave a significantly larger
residual mean square compared with either of the previous models. These results supported
the model described above that the differences between the TVB results obtained with the
different methods, other than the Stansby et al. (1944) procedure, were in the initial values
and were due to different amounts of decomposition ammonia. The lowest values of a were
7.4 and 8.7mgN (100g)-1
for the Conway and the distillation at reduced pressure methods.
Botta et al. (1984) do not describe how results were calculated, but presumably they were on
a mass ratio basis and these results, based on extracts would be slight overestimates
compared to calculation on a volume basis. The two highest values were obtained by
distillation of fish muscle with MgO; 18.1 mgN (100g)-1
by the L&G procedure, 15.3 by
rapid steam distillation. The distillation time in the former method is 20 minutes compared
with 10 minutes or less in the latter and the difference can be accounted for by the additional
decomposition with the longer distillation time. The initial value by distillation of a TCA
extract with NaOH was 12.8 mgN (100g)-1
.
Regulations in the European Union (EU) have set limits to TVB concentrations for some
species of fish and the recommended method of analysis, steam distillation in a rapid
distillation unit of a PCA extract at a ratio of 1:9, muscle:extractant, made alkaline with
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18
NaOH, is set out in an EU Regulation (European Commission, 2005). The Regulation though
allows for other procedures to be used and Vyncke (1996) compared the results obtained by
the recommended procedure with those alternatives. He found a very high correlation
between the procedures and presents figures for results of four pairs of them along with the
regression equations. In all cases the regression coefficients are close to 1.0 and the intercepts
close to 0.0, but the author does not list the errors of the parameters. The reviewer has further
analysed the results by reading data from the figures and calculating the structural
relationships and confidence bounds of the parameters. The results included some very high
TVB concentrations and in view of the findings discussed above that the amount of
decomposition ammonia might be greater in very stale fish compared with fresh the statistical
analyses were restricted to comparisons in which TVB contents were less than 50 mgN
(100g)-1
. Vyncke (1996) used a Kjeltec Tecator unit for distillation of an extract according to
the recommended method and compared the results with distillation in an Antona unit
(Antonacopoulos, 1960). One set of data, Figure 1 in the paper, compared distillation of PCA
extracts, the EU recommended procedure. The structural relationship of TVB contents using
the Antona apparatus on TVB contents using the Tecator has a slope of 1.085 with 95%
Confidence Limits (CL) of 0.96 and 1.22, that is, including 1.0. When the structural
relationship is forced to have a slope of 1.0, the intercept is 2.93, that is, TVB determined
using the Altona apparatus gives a value almost 3mgN (100g)-1
larger than when using the
recommended EU procedure with the Tecator unit. Vyncke (1996) records that the distillation
time using the Tecator unit was six minutes compared with 12 minutes using the Antona
apparatus. As was discussed above decomposition ammonia is produced during distillation of
protein-free extracts with NaOH and the cause of the bias of the Altona over the Tecator
procedures can be attributed to the extra distillation time in the Altona procedure. The EU
Regulation permits direct distillation of muscle alkalised with MgO in the Altona apparatus
and TVB results by this procedure were compared with those using the Tecator unit. The
structural relationship of Altona results on Tecator had a slope of 0.967 with 95% CL of 0.85
and 1.10, that is, again including 1.0. Forcing a slope of 1.0 on the structural relationship
gives an intercept of 2.97. Again, bearing in mind the earlier discussion of the rate of
formation of decomposition ammonia by distillation of muscle with MgO, this bias of the
Altona over the Tecator can be accounted for by the longer distillation time when using the
Altona apparatus. The direct distillation in the Altona apparatus was also compared with
distillation of the corresponding PCA extract alkalised with NaOH and distilled in the Antona
apparatus. The paper, Vyncke (1996), does not describe the details of the extraction
procedure and the calculation of concentration, but presumably the procedure described in the
EU Regulation was used. This, as discussed above, does not allow for the water content of
the sample and would overestimate the TVB content derived from the PCA extraction by a
factor of 1.02. When this correction is applied to the original data the structural relationship
of direct distillation of muscle on distillation of PCA extract has a slope of 1.037 with 95%
CL of 0.951 and 1.132. Forcing the structural relationship to have a slope of 1.0 gives an
intercept of -1.38, that is direct distillation with MgO gives a TVB content 1.38mgN (100g)-1
higher than the distillation of a PCA extract does. The distillation conditions of the two
methods are the same so this bias most likely can be attributed to a difference in the amount
of decomposition ammonia produced. The fourth comparison in Vyncke (1996) is PCA
extract as specified in the EU recommended method against a 1:3 fish:extractant in TCA,
(Codex method), which is specified in the Regulation as an alternative. Again, as discussed
above, the TVB content in the sample would be calculated on the mass ratio and would
overestimate the TVB content on a flesh basis. After correcting values as given in the paper,
Figure 4, for both extractants the slope of the structural relationship of TCA on PCA results
was 1.097 with 95% CL of .975 and 1.235. When the structural relationship is forced to a
slope of 1.0 the intercept is 0.38 with 95% CL of -0.12 to 0.89, that is includes a value of 0.0.
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It appears from this Vyncke (1996) study that equation (4) above is a good model for the
results with c depending on the material being distilled B intact muscle tissue or extract B and
the distillation time.
B. TMA
1. The Conway Method
In the early decades of the last century TMA was determined by tedious analytical procedures
based on separate measurement of ammonia and selective decomposition of the amines with
nitrous acid in a TVB distillate (Weber and Wilson,1918; Okoloff, 1932; Boury and Schvinte,
1935; Hjorth-Hansen and Bakken, 1947). Many of the analytical procedures introduced since
then have not been specific for TMA, but have used formaldehyde to suppress interference
from ammonia and methylamines other than TMA. Beatty and Gibbons (1937) introduced a
simple method of analysis using the Conway microdiffusion cell (Conway 1950) in which
1ml of press juice, the liquor obtained from minced fish muscle by mechanical pressure, is
mixed with 0.5ml of formalin solution in the annular ring of the cell, the mixture made
alkaline with saturated potassium carbonate and the liberated bases absorbed in standard acid
in the central well. The excess acid is back titrated as usual to give the amount of bases
distilled over. Under the experimental conditions specified the ammonia is complexed and
does not distil over, but DMA is probably not completely, or at all, complexed and the
method is probably not specific for TMA as Beatty and Gibbons (1937) themselves point out.
The Beatty and Gibbons method for determination of 'TMA' is operationally the same as that
of the Conway method for measuring TVB and has the same advantages and disadvantages as
already discussed for the latter determination and if TVB is being measured by the
microdiffusion procedure it is convenient to measure the former as well. Though many other
procedures for determination of TMA have been described since Beatty and Gibbons (1937)
the microdiffusion method is still in use; the reviewer is aware of at least 10 publications in
research journals in or since 2000 which report its use.
2. The Picrate Method
Dyer (1945) described a colorimetric method based on a simplified version of one proposed
for determination of amines in blood (Richter et al., 1941). The principle of the Dyer (1945)
procedure is that an aliquot of an extract of fish muscle is taken, formaldehyde added to
prevent interference by ammonia, and the mixture made alkaline. The free bases are extracted
into solvent, usually toluene, and reacted with picric acid to give a yellow-coloured picrate
salt which can be measured in a photometer. The materials, equipment and expertise required
for the picrate method are available in any food analytical laboratory and the procedure is
suitable for use commercial and regulatory laboratories as well as in research. The most
expensive item is a photometer, but a reasonably accurate estimate of optical density can be
achieved by comparing the test sample against a suitable range of standards. Though the
procedure for determination of TMA by the picrate method is quite straightforward some
precautions must be taken to ensure accurate and precise results. The original procedure
(Dyer, 1945) used saturated potassium carbonate as the alkaliser, but the combination of
formaldehyde with potassium carbonate does not completely prevent partition of DMA into
the solvent phase. Further studies showed that potassium hydroxide (KOH) was more
efficient in suppressing interference from DMA and in giving higher recoveries of TMA
(Hashimoto and Okaichi, 1957; Shewan et al., 1971; Tozawa et al., 1971; Keay and Hardy,
1972; Bullard and Collins,1980), but the there are other factors as well. The amines are not
completely partitioned into the solvent phase and the fraction of TMA extracted depends on
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the composition of the alkaliser. For example, Bullard and Collins (1980) found that 97% of
the TMA was extracted into the toluene phase using 45% KOH, 80% using 25% KOH and
72% using 50% potassium carbonate. The effects of the different alkalisers is almost certainly
a salting out effect, not an effect of alkalinity. They also found that none of the
alkali/formaldehyde combinations completely suppressed the interference of DMA. 25%
KOH gave the least interference of DMA with a colour yield for DMA relative to TMA of
about 6% compared with about 13% for 45%KOH. Wong and Gill (1987) reported that the
picrate procedure using 25% KOH overestimated TMA content compared with an High
Performance Liquid Chromatography (HPLC) procedure and Prez-Villarreal and Howgate
(1986) reported that the picrate procedure using 45%KOH overestimated TMA by 20%
compared with a GLC procedure. It appears from the various studies of the effect of the
nature and concentration of alkali in the picrate procedure that 45% KOH should be used in
situations where concentrations of DMA are expected to be low compared with TMA, for
example in chill-stored fish, because this gives a more complete recovery of the TMA and the
procedure is more sensitive, but where DMA is expected to be high, for example in frozen-
stored fish, 50% potassium carbonate should be used to reduce interference from DMA at the
expense of some loss of sensitivity to TMA.
Judging from publications in research journals when the picrate procedure is used in studies
of TMA content in fishery products the practice is about evenly divided between using
potassium carbonate or KOH as the alkaliser. It is perhaps worth noting that the method for
'Trimethylamine nitrogen in seafood' recommended in the AOAC Official Methods of
Analysis specifies saturated potassium carbonate as the alkaliser, whereas the procedure
specified by the Analytical Methods Committee uses 45% KOH
It is usual to standardise the picrate procedure for each batch of analyses by preparing a
calibration curve at the time using standard solutions of TMA. Bearing in mind that
extraction of TMA into the solvent phase is not complete, especially when using 25% KOH
or potassium carbonate, it is important that the working standards be made up in a solution
that replicates the solute contents of test extracts. For example if a 1:3, sample:extractant,
extract is prepared in 10% TCA and the sample is 80% water then the working standards
should be made up in 8% TCA.
Formaldehyde in near neutral solutions reacts with salts of ammonia and MMA to release
equimolar amounts of acid and this reaction can be utilised to estimate TMA by an extension
of the TVB procedure in which formaldehyde is added to the neutralised distillate from a
TVB determination and titrating further the acid released (Boury and Schvinte, 1935;
Analytical Methods Committee, 1979). It is often assumed in accounts of the reactions of
formaldehyde with DMA that a similar reaction occurs with this amine as well, but according
to Boury and Schvinte (1935) formaldehyde reacts with DMA salts, but does not release acid.
The difference between the titrations of a TVB distillate before and after addition of
formaldehyde therefore estimates the amounts of DMA and TMA. However, DMA is usually
a minor component of the volatile bases in spoiling fish and the procedure would usually
provide an adequate estimate of TMA content for many purpose in quality control of fishery
products. The titrations need to carried out with care to give accurate results. The volumes of
solution before addition of formaldehyde should be kept as small as possible and carbonate-
free alkali should be used in the titrations. A pH indicator changing colour at pH 7.0 should
be used and it might be useful to have a buffer solution at pH 7.0 with the indicator added as
a reference to match the end points.
3. Distillation with Formaldehyde
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If it is assumed that formaldehyde fixes ammonia and methylamines other than TMA then it
would seem reasonable that adding formaldehyde to the distilling mix in the determination of
TVB should allow only TMA to distill over. Beatty and Gibbons (1937) refer to trying
distillation in the presence of formaldehyde as a procedure for measuring TMA in fish, but do
not give any results of their trials. The principle was investigated by Benoit and Norris (1942)
who, in the introduction to their paper, cited publications going back to the end of the 19th
century which had used distillation in the presence of formaldehyde as a means of separating
mixtures of methylamines. Benoit and Norris (1942) distilled solutions of ammonia and
methylamines with formaldehyde under reduced pressure at 301C using sodium carbonate as
the alkaliser. They showed that TMA was quantitatively recovered at all concentrations of
formaldehyde used and that ammonia did not distill over. The behaviours of DMA and MMA
were anomalous in that at low concentrations of formaldehyde distillation of these bases was
partially suppressed, but as its concentration was increased a higher proportion of these bases
distilled over. The procedure then is not specific for TMA B though they used it in a study of
TMAO content of various species of aquatic animals (Norris and Benoit, 1945) B and the
reviewer is not aware of the procedure being otherwise used for determination of TMA in
fishery products. Malle and Tao (1987) later revived the principle of the Benoit and Norris
(1942) procedure, (though without citing that or other papers), by distilling a TCA extract
with formaldehyde at atmospheric pressure using sodium hydroxide as the alkaliser. They do
not report recoveries from model solutions of methylamines, but compared TMA values of
fish samples obtained by their procedure with those obtained by the microdiffusion or the
picrate procedures. There is a high correlation between the two sets of data, but fitting a
structural relationship to the results reproduced in the paper shows that the Malle and Tao
procedure overestimates the TMA content compared with the microdiffusion procedure by
about 22% over all the samples. The reviewer's own experience (unpublished) of the Malle
and Tao method with solutions of ammonium chloride shows that the ammonia is not
completely fixed and the proportion distilling over is very dependent on the alkalinity of the
distilling mix. Even at low alkalinities, (but sufficient to allow for rapid distillation of
amines), about 15% of the ammonia comes over and at the alkalinity expected from the
amounts of extract and alkali used by Malle and Tao (1987) 25% distilled over. Though the
amine/formaldehyde reactions summarised in the paper occur in approximately neutral
solutions at room temperature the reactions occurring under the distillation conditions of the
Malle and Tao (1987) procedure will be more complex. The formaldehyde would decompose
under the hot alkaline conditions by the Cannizarro reaction at least and lose its effectiveness,
and decomposition products would undergo the Mannich reaction with the dimethylamine
with the possible release of other amines.
4. Gas Liquid Chromatography
Amines were amongst the first groups of compounds to be separated by gas liquid
chromatography (GLC) when the technique was introduced (James et al., 1952) and was soon
applied to the determination of amines in fishery products (Groninger, 1958; Hughes, 1958;
Hughes,1959). Since then a variety of analytical procedures and column packings for
effecting the separation have been described in the literature of determining amines in fishery
products. Amines tend to 'tail' and variations in column packings tend to be directed towards
reducing this effect. A more fundamental difference in procedures is in the treatment of the
sample before injection on the column. Earlier procedures (Groninger, 1958; Gruger, 1962;
Hughes, 1958; Hughes,1959) injected the distillate from a TVB-type distillation directly on to
the column. This approach has the disadvantage that injection of water onto the columns
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usually has an adverse effect on their performance. This can be avoided by extracting the
amines from an aqueous extract into an organic solvent and injecting an aliquot of that
(Nonaka, et al., 1967; Keay and Hardy, 1972; Tokunaga, et al., 1977; Lundstrom and
Racicot, 1983; Perez-Martin, et al., 1987; Krzymien and Elias, 1990; Oetjen and Karl, 1999).
The effects of incomplete partition of the amines into the solvent already discussed above
with regard to the picrate picrate method apply to this procedure as well and recoveries of
amines using different solvents are measured and discussed in Lundstrom and Racicot,
(1983). The analysis of headspace vapours above an alkalinised suspension or extract of a
sample avoids the need for a distillation or extraction step (Miller, et al., 1972; Kruse and
Stockemer, 1989; Krzymien and Elias, 1990; Fiddler, et al., 1991). The headspace of course
will contain volatiles other than amines and Miller, et al., (1972) used a nitrogen detector to
reduce interference from non-nitrogen-containing compounds, but Kruse and Stockemer,
(1989) and Fiddler, et al., (1991) in comparing the nitrogen detector with standard FID found
the latter was satisfactory. An extension of the simple head space sampling is to use solid-
phase microextraction (Bn et al., 2001; Chan et al., 2006). Chan et al. (2006) used their
procedure to measure TMA in two species of freshwater fish and found high, >30 mgN
(100g)-1
, in chill-stored samples, which is very surprising bearing in mind that generally
freshw