Chapter 1

Introduction and Literature

Review
 Chapter -1

Introduction and Literature Review

 Chapter 1

1. Grape

1.1 The Origin and Evolution

Grape is believed to have originated in Armenia near the Black and Caspian
seas in Russia. An independent and recent origin of grapes is also traced to North
America. From Armenia, grapes spread westwards to Europe and Eastwards to Iran
and Afghanistan. Grape was introduced into India in 1300 AD by the Moghul
invaders. Grape cultivation flourished in Baluchistan and North-West Frontier
Province during the 16th century. In India, grape cultivation declined after the fall of
Moghul rulers but was reintroduced in south India (Aurangabad district of
Maharashtra) by Mohammed-Bin-Tughlak and since last 60 years grape is
commercially cultivated in India. The old Vitis vinifera grapes, originating in
Armenia, have perfect flowers while the grapes of America, which are of recent
origin, usually have imperfect flowers. It is believed that originally varieties with
pure male / female flowers to varieties with various degrees of maleness / femaleness
to those with perfect flowers existed and during the course of evolution only the
varieties with perfect flowers have been selected.

1.2 Reproductive Biology of Grape

The flowers of cultivated grapes are usually hermaphrodite (perfect), while

wild grapes are often dioecious. Flower buds just before bloom (anthesis) are
covered by the interlocking petals (cap or calyptra). At anthesis the cap separates
from the base of the ovary and falls off. The stamens spread out and pollen is shed
and falls on to the stigma of the pistil. This is bloom and it lasts from 2-7 days
depending on temperature. In grapes not all ovules are capable of fertilization and
the unfertilized ovules drop off. This is known as shatter. Commercial grapes mostly
belong to Euvitis section comprising of V. vinifera, V. labrusca, V. riparia and V.
rupestris with the haploid chromosome number 19. In the other section, Muscadinia,
the haploid chromosome number is 20. The species of grapes are quite heterozygous
and seedling offspring show wide genetic variability. Seedlings vary not only in the
qualities of their fruit but in vegetative vigor also. Because of these variations, seeds
are not used for propagation of vines for vineyard purpose.

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1.3 Area, Production and Productivity

1.3.1. India on world background

Grapes are grown commercially in 89 countries worldwide. Table 1.1
narrates the present status of area, production and yield of grapes in India with
reference to world and leading countries during the year 2007-08. Total area under
grape was 7.5 million hectare with total production of 66.3 million MT of fresh
grapes. Spain stands first with 16.00% of total area under grape cultivation while
Italy aheads with 12.85% share in world total fresh grape production. India accounts
for 64.4 thousand hectare area (0.86%) and 1.68 million MT (2.53%) of grape
production. In terms of yield per hectare, India stands first with average fresh grape
production of 26.00 MT/ha as against world average 8.84 MT/ha.

Table 1.1: Present Status of Area, Production and Yield of Grapes in India with
Reference to World and other Leading Countries during the Year 2007-08

World India % Share Leading countries in the world

of India
Name Qty MT % Share
Grape area 7,503,000 64,400 0.86 Spain 1,200,000 16.00
of harvest
France 830,000 11.06
(Ha)
Italy 770,000 10.26
Grape 66,325,000 1,677,100 2.53 Italy 8519418 12.85
production
France 6500000 9.80
(tonnes)
China 6250000 9.40
Grape 8.84 26.00 India 26.00
yield
USA 16.07
(tonnes/ha)
South Africa 13.91

(Ref: FAOSTAT 2009, Indian Horticulture Database 2008)

Table 1.2 presents export-import of grapes and various grape products at

global level vis-à-vis India during 2006-07. In spite of the highest productivity in
India, it lags far behind in terms of share in export of fresh grapes (2.50%), raisins
(0.04%) and wines (0.01%). With reference to global trade Chile leads in fresh grape
export (19.23%) while France leads in wine export (34.88%).

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Table 1.2: Export-import of Various Grape Products at Global Level vis-à-vis India
during the Year 2006-07

Product World India % Share Leading countries in the World

trade (MT) (MT) Qty Name Qty (MT) % Share
(Value)
Export 3,416,087 85,563 2.50 Chile 823,198 24.09 (19.23)
of (1.65) Italy 417,217 12.21 (12.41)
grapes USA 290,008 8.48 (12.36)
Export 774,502 355 0.04 Turkey 240,743 31.08 (28.13)
of (0.03) Iran 148,035 19.11 (13.24)
raisins USA 113,897 14.71 (21.58)
Export 8,352,554 1,059 0.01 France 1,461,663 17.50 (34.88)
of (0.01) Italy 1,793,152 21.47 (18.01)
wine Spain 1,336,762 16.00 ( 8.74)
Import 3,387,567 1,976 0.06 USA 530,889 15.67 (18.08)
of (0.04) Germany 326,546 9.64 (9.89)
grapes Russia 320,677 9.47 (6.73)
Import 8,874 1.13 UK 117,569 16.62
of (1.34) Germany 79,404 10.22
raisins Russia 67,910 4.19
Import 7,808,225 1,815 0.02 Germany 1,330,423 17.04 (10.58)
of (0.05) UK 1,184,626 15.17 (18.38)
wine USA 782,423 10.02 (18.42)

(Ref: FAOSTAT 2009, Indian Horticulture Database 2008)

1.3.2 Maharashtra on India background

The figures 1a and 1b illustrate the state wise area and production of grape in
India during 2007-08. The state of Maharashtra stands at leading position with 46.60
thousand hectare area with 72.36% share in total area under grape cultivation in
India. Maharashtra produced 1.29 million MT fresh grapes with 76.92% share in
India. Karnataka (12.46%), Tamilnadu (4.98%) and Andhra Pradesh (3.12%) were
other major grape growing states.

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 Chapter 1

A. 3.82%
0.03%
0.15%
1.53%
0.15%
0.28%
4.28%
0.31%
1.07%
16.97%

0.15%

71.25%

B. 3.12%
0.01%
0.17%
1.59%
0.03%
0.01% 0.01%
4.98%
0.49% 12.46%

0.21%

76.92%
Andhra Pradesh Haryana Himachal Pradesh Jammu and Kashmir
Karnataka Madhya Pradesh Maharashtra Mizoram
Nagaland Punjab Rajasthan Tamilnadu

(Ref: Indian Horticulture Database, 2008)

Figure 1.1 State wise area (A) and production (B) of Grapes in India during the Year
2007-08.

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 Chapter 1

1.3.3 Consumption Pattern

There is vast difference in consumption pattern of grapes in India as
compared to other major grape growing countries of the world. The major food
products made from grapes are reflected in the utilization data (USDA 2008).

Table 1.3: Consumption pattern of grapes in India.

Particulars India World

Table grapes 75-80% 10-15%
Raisins (dried grapes) 17-20% 25-30%
Wine 0.5% 50-55%
Juice, Jelly, Canned foods 1.5% 6-10%

Currently 75-80% production is utilized as table grapes. The major bulk is harvested
in March-April, but as cold storage facilities are inadequate, there are frequent
market gluts. Therefore, there is need to diversify the uses of grapes. There is great
scope for development of grape wine industry in India. Looking forward to this
Maharashtra and Karnataka State Governments has come up with new policies for
promoting grape processing.

Grape is an important commercial fruit crop, which receives frequent

application of large number of agrochemicals, e.g. pesticides etc. throughout the
cropping season for management of various pests and diseases. At present, in India
grape is grown over an area of 60,000 ha with an annual production of 1.2 million
tonnes (FAO, 2005). Although exact figures are not available regarding the current
area and production of wine grapes in India, it is estimated that around 1200 hectares
with annual Production of 9.5milian liters. In Maharashtra and about 100 hectares
near Bangalore in Karnataka are currently under wine grape cultivation. Indian grape
is under constant scrutiny of the environment and health protection agencies
worldwide, as in India, the cultivation of grapes receives frequent application of
large number of pesticides and further, grape is mostly consumed as fresh fruit in
intact form without any processing. The residues left on the grapes during harvest
can be carried through into the wine [1].

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1.4 Residue Analysis Literature Review

In food analysis, the methods of analysis need to confirm and quantify as

many residues as possible. The multi-analysis of pesticides is a difficult task for the
chemist, due to the variety of different groups of pesticides having a broad range of
physio-chemical properties, e.g. polarity, thermal stability, acidity etc. Determination
of pesticide residues in food are often complicated by the presence of fats and
interfering natural compounds, this is why it is often requires a time-consuming
clean up and pre-concentration procedure before analysis. Due to the large number
of pesticides on the world market, the development of multi-residue methods is
[2]
preferred in terms of pesticide residue analysis . Food matrices are complex and
may be difficult to analyze and require highly selective analytical techniques to
determine target compounds and to characterize unknown compounds. The use of
Gas Chromatography (GC) and Liquid Chromatography (LC) with Mass
Spectrometry (MS) is a well-accepted method for confirmation on the identity of
pesticides at very high sensitivity, whereas Inductively Coupled Plasma-Mass
Spectrometry (ICP-MS) has become an analytical technique to determine the content
of inorganic compounds in food. Although LC-MS-MS is a powerful tool for fast
and selective analysis, which enables chromatography with low separation
efficiency, coeluting compounds can lead to problems in the MS response due to ion
suppression of the signal. So an efficient sample purification and chromatographic
separation should not be under-evaluated [3].

The interest for making the analysis as quick and cheap as possible have led
scientists to search for a coupling between the clean-up system and the system for
the determination of analytes, and also to reduce the clean-up/concentration
procedure. Slobodník et al. [4] have used on-line coupling of solid phase extraction
(SPE)-LC to MS to detect 17 pesticides in water and trace enrichment separation
[5]
before LC-MS was used by Hogenboom et al. for the determination of 17
pesticides in water.

The use of pesticides to control pests of crops, to eliminate parasites and

insects of livestock, and to maintain hygienic conditions in production lines or
during storage, has played a major role in expanding the availability of produce to
the consumer because they have allowed growers and handlers to increase

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production volume, extend shelf life, and improve the appearance of many foods.
The consequences of their use and the realization that some foods contain residues of
these compounds are of paramount importance to the consumers. Such contaminated
food can readily reach the population and growing concern has been expressed as to
the possible hazards for human health. Since human safety is the foremost
consideration in food production, superseding even the obvious importance of
economic factors, international systems of legal control have been established to
prevent residue contaminated products from entering the human food supply. In this
way, an important aspect of food safety is the control of pesticide residues on food

The analysis of pesticide residues represents a basic instrument not only for
the protection of human health, through risk assessment studies and prevention
strategies, but also for trade and official control purposes. There is a growing interest
to apply liquid chromatography mass spectrometry (LC-MS) in control of pesticide
residues to ensure food safety. Social Sciences Citation Index, and Arts &
Humanities Citation Index) using the keywords ‘‘Pesticides, Food,’’ and ‘‘Liquid
Chromatography-Mass Spectrometry.’’ Among these articles, there are several
reviews, which have been published since 2000, about some aspects of pesticide
residue control by LC-MS, as applications of some of these instruments in pesticide
trace analysis or as representative with other techniques used in the analysis of
pesticide residues in food and drinks.

This review addresses exclusively LC-MS and is thus focused on the LC-MS
analysis of pesticide residues to ensure food safety. After an introduction about the
regulations that highlights the importance of these techniques to meet the official
requirements on analytical performance, the different mass spectrometers used in
this field of research, as well as the LCMS interfaces and the difficulties associated
with quantitative LC-MS determination, are discussed. The ability to use practical
data for quantifying pesticides together with the option of obtaining structural
information to identify target and non-target parent compounds and metabolites is
illustrated. Special attention is paid to the impact of sample preparation and
chromatography on the ionization efficiency of pesticides from food. The last section
is devoted to its applications from a food safety point of view. [6]

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1.5 Pesticides and Health

Public concern over pesticide residues in food is increasing since it has

become a significant food safety concern. Very little data are available regarding
human exposure to pesticides through consumption of processed and finished food
products. However, a study by Andrey and Amstutz[7] showed that 61% of 83
labeled “organic” wines and 87% of 15 conventional wines found in Swiss market
places contained pesticide residues. Currently, there are few studies in the United
States on the presence of pesticides in wines or alcohol-based beverage products,
although there are tolerances set for table and wine grapes[8,9]. These concerns have
caused many regulatory agencies to increase their scope of analysis as well as the
number of samples analyzed in their monitoring programs for risk assessment[9] .
Cabras et al[10] and Navarro et al[12, 13, 14] explored into the fate of pesticides from the
processed grapes through the vinification process to the final wine product. There
were many multiresidue procedure for beverage alcohol products such as wine[11]

1.5.1 Regulations

Maximum residue limits (MRLs): Currently, more than 800 pesticides (active
ingredients) are sold worldwide. For many of these compounds, legal action levels
(e.g., maximum residue levels (MRLs) or tolerances) in food have been established.
Pesticide residues have been regulated by several legislative authorities throughout
the world, all concerned with the quality, efficacy, and safety in the use of pesticides.
For instance, MLRs for pesticide residues are set at European Union (EU) level for
approximately 150 plant protection products and at member state level for any other
unharmonized products. In an attempt to facilitate world trade while at the same time
protecting the health of the consumers, the Food and Agriculture Organization
(FAO) and the World Health Organization (WHO) of the United Nations (UN) have
set a joint FAO/WHO Codex Alimentarius Commission to coordinate food
standards. One of the main tasks of the Codex committee is to establish universal
MRLs [15].

EU has also concerned with the concordance of pertinent regulations that

differ among member states to allow the free circulation in the market of food.
Considerable progress has been made in establishing EU-harmonized MRLs for all

8
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pesticide residues. New laws, such as the European Directive 91/414/EEC [16] or the
[17]
Food Quality Protection Act (FQPA) in the United States of America (USA) ,
have increased the standards for human health. The quality standards include the
reassessment of the MRLs, which have been reduced in comparison to the previous
[18, 19, 20]
limits . In general, the MRLs are in the range of 0.01–10 mg/kg, depending
on the combination commodity and pesticide, the lowest is characteristic of banned
compounds because it is considered that this would be the minimum limit of
detection (LODs) achievable. A value of zero is considered below the LOD because
of the slight inaccuracies in the measurement methods available.

These regulations encompass special provisions for infants and children,

including additional safety factors. Specific rules on the presence of pesticide
residues in infant and follow-on formulae, as well as in processed cereal-based baby
food and baby food are set out in Commission Directives 1999/50/EC and
[21,22]
1999/39/EC . These Directives require that baby food contains no detectable
levels of pesticide residues, meaning not more than 0.01 mg/kg of pesticide residues.
In addition, Directives 2003/14/EC and 2003/13/EC prohibit the use of certain very
toxic pesticides in the production of any baby food, and establish levels lower than
the general maximum level of 0.01 mg/kg for a few other very toxic pesticides [23, 24].

1.6 Grape and Wine Chemistry

In the recent years, viticulture and enology play an important role for
economy of many countries, and considerable efforts are devoted to improve the
product quality and to match the widest approval of market. Many important
industrial processes are finalized to improve organoleptic characteristics of wine:
alcoholic fermentation is promoted by inoculums of selected yeast, extraction of
grape components is enhanced by maceration of grape skins in controlled conditions
and by addition of selected enzymes, malolactic fermentation and barrel- and bottle-
aging are performed to achieve biological stability and to improve flavor and
[25]
fragrance of product . To guarantee the quality of product, all these steps have to
be monitored and verified. Community laws, as well as the single country ones, are
devoted to protect the consumer health, other than the internal market from
introduction of low quality products, by accurate controls of foods.

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As a consequence, for exporting of wine and derivate products, quality

certificates are often required, in particular with regard to the presence of
contaminants such as pesticides, heavy metals, ethyl carbamate and toxins, for which
legal limits are often defined. To prevent frauds and to confirm the product identity,
accordance between the real product characteristics and the producer declarations
(e.g., variety, geographic origin, quality and vintage) has to be verified. Some
maximum limits of grape and wine contaminants are fixed by national and
community regulations, and are reported in Table 1.3

Table 1.4: Maximum Limits of Some Grape and Wine Contaminants Fixed by
Regulations of Single Countries, European Union(EU), United Nations (UN)

Contaminant Grape Wine grape Country Source

(mg/kg) (ppm) juice
(ppm)
Carbaryl 3 -- -- Italy DM 14.12.2004
5 -- -- UN Codex alimentarius
Diuron 0.05 -- -- Italy DM 14.12.2004
Fenoxycarb 0.02 -- -- Italy DM 14.12.2004
Folpet 10 -- -- Italy DM 14.12.2004
2 -- -- UN Codex alimentarius
Iprodione 10 2* -- UN / Italy Codex alimentarius/
DM14.12.2004
Myclobutanil 1 0.1* -- UN / Italy Codex alimentarius/
DM14.12.2004
Penconazole 0.2 -- -- UN / Italy Codex alimentarius/
DM14.12.2004
Primicarb 0.2 -- -- Italy DM 14.12.2004
Procymidone 5 0.5 -- Italy Codex alimentarius/
DM14.12.2004
Propiconazole 0.5 -- -- Italy DM 14.12.2004
Triadimefon 2 -- -- Italy DM 14.12.2004
Vinclozolin 5 -- -- UN / Italy Codex alimentarius/
DM14.12.2004
Ochrotoxin A 0.002 0.002 0.002 EU CE regulation no 123/2005
Pb 0.2 0.2 0.05 EU CE regulation no 466/2001
Histamine -- 2 Germany Recommended (souza et
al., 2005)
- 5 Belgium Recommended (souza et
al.,2005)
8 France Recommended (souza et
al.,2005)
10 Switzerland Recommended (souza et
al.,2005)
*Only for Italy.

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Activity of researchers and organisms of control is devoted to develop new methods

[26],
to verify the product origin to detect illegal additions and adulteration such as
sugarbeet, cane sugar or ethanol addition and watering[27], to protect consumer health
through determination of food contaminants[28-31].

GC/MS and LC/MS have also been applied to develop methods for the legal
parameters control finalized to the consumer health protection and to prevent frauds,
such as determination of pesticides in wine, detection of compounds formed during
alcoholic fermentation by yeast and bacteria, determination of illegal additions to the
wine. Also methods for determination of toxins in the wine have been proposed [32].
Inductively coupled plasma-mass spectrometry (ICP/MS) is nowadays a large-used
technique to determine heavy metals in wine. For these aims the knowledge of the
chemical composition of grape and wine is essential.

There is a large interest regarding health and safety issues associated with the
fungicides, insecticides and herbicide use, and the possible presence of their residues
in processed foods and drinks. The high concern about health risks connected with
pesticides, led to the development of several European Community (EC) Directives
(also adopted by the Italian Legislation) stating maximum residue limits (MRLs)
tolerated for each food commodity[33,34]. Wine and grape are included among these
commodities, in particular in wine the procymidone MRL has been defined to 0.5
mg/L, such as for cyprodinil and fludioxomil, 0.1 mg/L for myclobutanil, and 2
mg/L for iprodione; MRLs in grape have been fixed to 10 mg/kg for folpet, 5 mg/kg
for vinclozolin, and 3 mg/kg for carbaryl [35].

Fungicides, insecticides, and herbicide are commonly used in viticulture.

Dicarboxyimide fungicides have been widely used against Botrytis cinerea in
vineyards. Vineyards are treated in the final stage of vegetation to prevent grape’s
attack, which may occurs shortly before the harvest. Among them, vinclozolin and
iprodione are currently employed in Italy[36,37].These fungicides show reduced
toxicity, but 3, 5-dichloroaniline,the probable common final product of their
degradation or metabolic pathway, seems to be as hazardous as other aromatic
amines. Although maximum residue limits for most pesticides in wine have not been

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fixed, several countries have established guidelines in the authorized use of

pesticides and MRLs for the treatment of vines and grapes used in wine production.
Pesticide residues are regulated at MRLs in grapes at low ppm levels. Because the
vinification process lowers the level of pesticides, their contents in wines are
significantly lower than in grapes. As a consequence, methods to detect pesticide
[38]
residues must be very effective and sensitive .One of the first MS methods for
determination of pesticides in wine reported in literature was GC/MS-EI by
performing analysis with a diphenyl-dimethyl polysiloxane capillary column and the
use of aldrin as internal standard (IS)[39] . Analysis of procymidone was performed
by liquid extraction of sample with hexane, and SIM mode analysis recording signals
at m/z 96 and 283 for procymidone, and m/z 263 and 265 for the IS. By using both
electronic capture detector (ECD) and MS detector, the same limit of quantification
(LOQ) of 2 mg/L was reported.

In 1996, analysis of vinclozolin and iprodione in wine by solid phase

extraction (SPE) sample preparation with a porous carbon stationary phase was
[37]
performed . Analytes were recovered with toluene and analyses performed by
GC/MS. Thermal stability, chemical resistance, and stability over a wide pH range of
carbon sorbents were evaluated. Recoveries of two fungicides in both standard
solutions and spiked wine samples ranging between 80% and 97% were reported.
MS analyses were carried out by ion trap detector (ITD) in both multiple ion
detection (MID) and SCAN mode. By selecting the characteristic ions of each
compounds, LOQs of 50 µg/L for vinclozolin (by recording signals at m/z 178, 180,
198, 200, 212, 215, 285, 287) and of 50 µg/L for iprodione (by recording signals at
m/z 187, 189, 244, 247, 314, 316), were recorded with a signal to noise ratio of 3
(S/N 3). By performing analysis of a vinclozolin 0.01 mg/L standard solution in MID
mode, unambiguous compound identification was obtained, by ITD sensitivity of
method for iprodione resulted significantly lower than for vinclozolin.

In the same year, a GC/MS method for analysis of fungicide metalaxyl in

wine by SPE sample preparation using a carbon sorbent, was performed
[40]
.Recoveries greater than 92% were reported for metalaxyl standard solutions at
concentration 3–100 mg/mL, whereas recoveries in spiked wines ranged from 80%
to 99% depending on the concentration and the sample matrix. LOQ by GC/MS-IT

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was 0.50 mg/L. Metalaxyl residue concentration in wine closely related to the
interval between the last treatment of the vines and the harvest of the grapes was
observed. Cabras et al. performed two different GC/MS methods by micro-extraction
with acetone/hexane to determine the fungicides cyprodinil, fludioxonil,
pyrimethanil, tebuconazole, azoxystrobin, fluazinam, kresoxim-methyl,
mepanipyrim, and tetraconazole in grapes, must, and wine [41] . The methods limits of
detection (LODs) resulted 0.05 mg/L for cyprodinil, pyrimethanil and kresoxim-
methyl, and of 0.10 mg/L for the other analytes [42,43].

To perform the routine monitoring of pesticides, in 1997 Kaufmann

developed a fully automated reverse-phase SPE and GC/MS method for the
[44]
simultaneous determination of 21 different pesticides in wine . By performing
SIM mode analysis and monitoring the m/z species, the method showed LODs
between 5 and 10 µg/L, and linearity regression coefficients greater than 0.99
(except for 4,4-dichloro-benzphenone and dicofol). Recoveries of 17 pesticides in
spiked wine samples ranged from 80% to 115%.In 1998, Vitali et al. proposed a
solid-phase micro-extraction (SPME) and GC/MS SIM mode method to determine
seven different insecticides (lindane, parathion, carbaryl, malathion, endosulfan,
methoxychlor, and methidathion), 4 fungicides (procymidone, folpet , vinclozoline,
and captan) and 3 herbicides (terbuthylazine, trifluralin and phosalone) in wine [45].

1.7 Method Development

Pesticide residue analysis of food and environmental samples has been

performed in numerous government and private laboratories throughout the world
for approximately 40 years. However, the methods used for analysis of common
pesticides are far from ideal. Some residue monitoring laboratories still use methods
developed 30 years ago when analytical needs were less demanding, solvent usage
was less of an issue, extended analysis time and manual labor were the norm, and
technology was less capable than today. Modern residue monitoring programs,
however, are expected to be responsive to the latest developments in agriculture and
new legislation.

The introduction of new, more rapid, and effective analytical approaches,

therefore, is essential for laboratories to improve overall analytical quality and

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laboratory efficiency. Without question, the most efficient approach to pesticide

analysis involves the use of multiclass, multiresidue methods (MRMs). The first
notable MRM was the Mills method developed in the 1960s by U.S. Food and Drug
Administration (FDA) chemist P.A. Mills [46]. At that time, non-polar organochlorine
insecticides (OCs) were the main focus for analysis. With the Mills method, OCs and
other non-polar pesticides were extracted from non-fatty foods with acetonitrile
(MeCN), which was then diluted with water, and the pesticides were partitioned into
a non-polar solvent (petroleum ether). As a consequence, relatively polar pesticides,
such as certain organophosphorus insecticides (OPs), were partially lost during this
step.

The need to analyze more polar OPs and other pesticides in agriculture
initiated the development of alternative procedures to determine compounds not
extracted by the Mills method. These methods often simply modified the Mills
procedure by using the initial acetonitrile extract but with different partitioning,
cleanup, and determinative steps [47-49]. In the 1970s, new methods were developed to
extend the analytical polarity range to cover OCs, OPs, and organonitrogen
[50,51]
pesticides (ONs) in a single procedure .These multiclass MRMs differed from
the Mills approach in that acetone, rather than acetonitrile, was used for the initial
extraction. However, the new methods still used non-polar solvents
(dichloromethane or dichloromethane–petroleum ether) to remove the water in a
liquid–liquid partitioning step.

Furthermore, NaCl was added to the water phase in both methods during the
partitioning step, the amount of which had a direct effect on the polarity range
covered by the methods. Becker[50], who developed the first MRM of this kind,
added an NaCl solution to the initial extract, which only partially saturated the water
phase with salt. Luke et al.[51] and Specht and Tilkes[52], however, added solid NaCl
to saturate the water phase, which forced more acetone into the organic layer, thus
increasing its polarity and leading to higher recoveries of the polar analytes. Soon
after their introductions, the Becker method became official in Germany as the DFG-
S8 procedure, and the Specht method became the official DFG-S19 procedure[53].
The Luke method, which replaced the Mills method in the FDA, became the official

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[54]
PAM 302 E1 procedure , and several years later became AOAC Official Method
[55,56]
985.22 .

These multiclass MRMs and their many variations are still widely used by
pesticide residue monitoring laboratories worldwide. Since the 1980s, environmental
and health concerns related to the use of chlorinated solvents have led to the
development of many new methods in which such solvents were avoided. Specht et
al.[57] and Anastassiades and Scherbaum[58] used a mixture of cyclohexane–ethyl
acetate (1 + 1) instead of dichloromethane (or dichloromethane–petroleum ether, 1 +
1) to induce partitioning. Casanova[59] and Nordenmeyer and Thier[60] used solid-
phase extraction (SPE) to extract pesticides from diluted acetone extracts, thus
completely avoiding liquid–liquid partitioning.

Luke et al.[61] added a combination of fructose,MgSO4, and NaCl to separate

water from acetone in the initial extract without using non-polar solvents. Schenck et
al.[62] also investigated the use of salts to form a water–acetone partition in extracts.
Parfitt[63] studied exposure of the initial Luke extracts to low temperatures to remove
the water phase by freezing it out of solution. All these separation approaches have
one or more disadvantages in practice because acetone is simply too miscible with
water to be easily separated without using non polar solvents. MeCN is more easily
and effectively separated from water than acetone[62-64] upon addition of salts. In the
case of MeCN, salt alone can be used to form a satisfactory separation from water,
and Lee et al.[65] used NaCl for this purpose rather than the nonpolar co-solvent of
the Mills method. Several other multiclass MRMs have been published since then
following this salting-out principle with MeCN[66-72] .

A third extraction solvent commonly used in multiresidue MRMs is ethyl

acetate (EtAc)[73-77]. EtAc has the advantage of partial immiscibility with water,
which makes the addition of other nonpolar solvents to separate water from the
extract unnecessary. However, a problem with EtAc is that some of the most polar
pesticides do not readily partition into the EtAc phase. To increase recoveries of
polar compounds, large amounts of Na2SO4 are usually added in MRM procedures,
with EtAc used to bind the water. Polar co-solvents, such as methanol and ethanol,
have been used to increase the polarity of the organic phase[76] . Another
disadvantage associated with the use of EtAc is the extraction of a large amount of

15
 Chapter 1

non polar co-extractives, such as lipids and epi-cuticular wax material, which must
be removed before the determinative step.

This is commonly achieved by a time-consuming and wasteful gel-

permeation chromatographic (GPC) cleanup step. During the 1990s, increased
urgency to further reduce solvent usage and manual labor in analytical laboratories
led to the commercial introduction of several alternative extraction approaches,
including supercritical fluid extraction (SFE; )[78-81], matrix solid-phase dispersion
(MSPD; )[82-84], microwave-assisted extraction (MAE; )[85], solid-phase micro-
extraction (SPME; )[86], and pressurized liquid extraction (PLE), also known
commercially as accelerated solvent extraction (ASE; )[87-89].Despite their
advantages, none of the techniques have overcome critical flaws or practical
limitations to enable their widespread implementation. For example, PLE and SFE,
which are instrument-based techniques that perform extractions in sequential, semi-
automated fashion, still require time-consuming manual steps and specialized items
that require cleaning after each use; sample throughput is not optimal, and
instruments and maintenance are costly.

SFE, MSPD, and SPME do not provide a wide enough analytical scope
within a single procedure, and MAE and PLE do not provide enough selectivity
(extracts require more cleanup than liquid-based extractions at room
temperature).Also, sample size in all of these approaches may be too small. These
approaches are useful for certain applications but are not sufficiently simple or
effective to provide the ideal MRM. Despite the many different multiclass MRMs
that have been described in the past 40 years, none has been truly streamlined to the
minimum factors that achieve a fast and easy extraction while still maintaining high
recoveries for a wide range of analytes and providing the selectivity and repeatability
needed of a reliable procedure. Many of the advantages and opportunities provided
by modern analytical techniques are not properly integrated into most current
methods. The aim of this study was to develop a simple, rapid, and inexpensive
multiclass MRM that provides high-quality results, but minimizes the number of
analytical steps, uses few reagents in small quantities, and requires very little
glassware. The main focus was to simplify the analytical process as much as possible

16
 Chapter 1

during extraction and cleanup without sacrificing high recoveries even for the most
difficult analytes.

Guidelines for Method Development:

Reliable residue analytical methods are necessary to measure the magnitude
of residues in a commodity, and to enforce legal MRLs (tolerances). That reliability
can be certified by two specifications. The most traditional one enforces the use of
standard methods. However, as a result of advances in analytical chemistry, the
concepts of routine methods and reference methods have been displaced in favor of a
‘‘criteria approach,’’ which establishes quality control principles, limits, and
[86-87]
procedures for the validation of screening and confirmatory methods . The
remarkable feature of this last tactic is its superior versatility, which allows the ready
adaptation of analytical methods to technical improvements and brings the
possibility to react rapidly to new emerging problems, such as, for example, in the
case of analyte/matrix combinations that have not been considered so far. The
analytical quality control (AQC) requirements are necessary to support the validity
of data used for checking MRLs, to support enforcement actions, or to assess
consumer exposure to pesticides. The objectives are (i) to ensure that false positives
or false negatives are not reported, (ii) to ensure that acceptable trueness (bias) and
precision are achieved, and (iii) to harmonize cost-effective AQC [86-87] . Laboratories
that carry out analysis of pesticide residues meet the requirements of a recognized
accreditation scheme, complying with ISO 17025 or Good Laboratory Practices
(GLPs). These approved laboratories prove their competence by regular and
successful participation in adequate proficiency testing schemes, recognized, or
organized by the national or community reference laboratories [86].

Both, the guidelines on quality control procedures for pesticide residue

[87]
analysis and the 2002/657/EC Commission Decision, concerning the
performance of analytical methods for the determination of organic residues and
contaminants in live animal and animal products [88] , state that ‘‘methods based only
on chromatographic analysis without the use of molecular spectrometric detection
are not suitable for use as confirmatory methods’’ and the latter explicitly requires a
combination of either an on-line and/or off-line chromatographic separation. In this

17
 Chapter 1

context, laboratories in many countries rely on detection by MS for unambiguous

confirmation of pesticides in food.

The guidelines on quality control procedures for pesticide residue analysis

establishes the principles of results confirmation indicating that when the increased
sensitivity obtained by scanning a limited mass range or by selected ion monitoring
(SIM) is essential, the minimum requirement is for data from two ions of m/z >200;
or three ions of m/z >100.Intensity ratios obtained from the more characteristic
isotopic ions may be of particular utility. If possible, the ions selected for
medium/high resolution mass spectrometry (MS) or tandem mass spectrometry
(MS/MS) should be characteristic of the analyte, not common to many organic
compounds.

The 2002/657/EC European Commission Decision also defines performance

requirements for the different approaches acceptable in mass spectrometric
detection: full mass spectra (full scans), SIM, tandem mass spectrometry (with
multiples stages) (MS/MSn) techniques, such as selected reaction monitoring (SRM)
and any other technique with appropriate ionization procedures. That Decision
outlines a recommended approach, using identification points (IPs). For the
confirmation of substances, a minimum of three or four IPs are required. According
to the report, any MS technique or combination of techniques may be employed to
attain the number of IPs needed for the identification of a compound. The number of
IPs achieved by MS detection depends on the technique used. The relative
intensities, both in full scan and in the SIM modes, of the detected ions, expressed as
a percentage of the most intense ion or transition, correspond to those of the
calibration standard within the tolerances. The smaller the relative ion intensity is,
the greater range of variations is permitted.

When mass spectrometric determination is performed by recording the full

spectra, the presence of all measured diagnostic ions (the molecular ion,
characteristic adducts of the molecular ion, and characteristic fragment ions and
isotopic ions), with a relative intensity of more than 10% in the reference spectrum
of the calibration standard, is mandatory. A minimum of four ions shall be present
with a relative intensity of >10%, including the molecular ion if possible. When
mass spectrometric determination is performed by SIM or using MS/MSn, the

18
 Chapter 1

molecular ion is preferably one of the selected diagnostic ions. These ions should not
be exclusively originated from the same part of the molecule. The signal-to-ion ratio
for each diagnostic ion is >3:1.These criteria are minimum performance criteria, that
is, a laboratory can decide to be more rigorous [88] .

1.9 Antibiotic Residues

At present, the occurrences of drug residues especially antibiotics in foods

and foodstuffs originating from veterinary uses have become increasingly apparent.
Sulfonamides (SAs) and tetracycline (TCs) are broad spectrum antibiotics frequently
used in Thailand as veterinary medicines. They are commonly used for the
prevention and treatment of dairy cattle for several infectious diseases, prophylactic,
or as feed additives to promote growth in farm animals [89].

An implementation of the effective monitoring program requires specific,

sensitive, and reliable analytical methods that can detect all drug residues below
these regulated levels. It is possible to analyze SAs, TCs, and PYR in foods.
[90, 91]
Microbiological and immunological assays are commonly used for rapid
screening. However, these techniques are complicated, lack sensitivity and
specificity, and thus are only suitable for semi-quantitative measurements. Liquid
[92-95] [96, 97]
chromatography (LC) coupled with UV , DAD and FLD emerged as
attractive alternatives for the determination of antibiotics due to their higher
[98] [99,100]
selectivity, sensitivity, and precision. GC and CE methods were also
reported. However, these techniques require preceding sample cleanup to achieve
acceptable detection. Currently, mass spectrometry (MS) technique has gained
popularity as a highly sensitive detection method for LC due to its ability to provide
simultaneous and unambiguous identification and quantification of drug residue at
trace levels.

From the literature review, we found that most of the published analytical
procedures were specific for the detection of each class of antibiotic. These methods
are varied in methodological approaches suitable for several varieties of matrices by
specific instrument. To the best of our knowledge, there is no report of an analytical
procedure that can separate and detect SAs, TCs, and Pyrimethamine (PYR) residues
simultaneously. This is due to the differences in structural and chemical properties of

19
 Chapter 1

these compounds that make simultaneous separation difficult. A detection of trace

level antibiotic residues in a rich matrix such as milk requires a good sample
preparation and cleanup procedure. Sample preparation procedures such as liquid–
[101]
liquid extraction (LLE) and solid-phase extraction (SPE) are commonly
employed for simultaneous extraction and cleanup.However, due to its ease of
operation and environmental interest, SPE has gained increasing popularity over
LLE. Common adsorbents including C8, C18, and ion-exchange have been applied
[102,]
for the analyses of SAs in various matrices such as milk and animal tissues
[102,103] [104-108]
. SPEs were also applied to the analyses of TCs in milk , TCs in
animal tissues [109-112].

Recently, an availability of dual quality polymeric SPE adsorbents such as

hydrophilic–lipophilic balance (HLB) made simultaneous enrichment and cleanup of
analytes in biological, environmental and food matrices possible. Oasis HLB SPE
cartridges were successfully applied for the analyses of SAs in honey , SAs in
wastewater [113-114], TCs in milk [115-116], and TCs in water [117-118]. There was a report
of simultaneous extraction and cleanup of SAs and TCs in environmental matrices
[119]
with Oasis HLB SPE cartridges as well . Simultaneous extraction of SAs and
PYR from human plasma was also well described [120-121]. However, there is a strong
need for a reliable simultaneous extraction and cleanup method for the analysis of
multi-class antibiotic residues in complex matrix such as milk and muscles for
routine monitoring purposes. A straightforward SPE cleanup and enrichment using
Oasis HLB SPE cartridges for six sulfonamides, three tetracyclines, and
pyrimethamine residues in milk was successfully developed in our laboratory
recently and will be reported elsewhere [122].

In order to ensure correct identification of the compounds, a reliable and

sensitive analytical method is also needed. This work describes a validation process
of the developed chromatographic and detection procedure coupled with the
developed simultaneous SPE sample preparation. Validation parameters tested
included method detection limit (MDL), method quantitation limit (MQL),
selectivity, linearity, precision, accuracy, and robustness. To enhance the quality and
accreditation of the method, the validation procedure was complied with acceptable

20
 Chapter 1
[123-124]
guidelines and used statistical techniques to guarantee the solidity of the
conclusion reached.

The major challenge when analyzing sediment is the extraction of the

antibiotic compounds from the solid phase to the liquid phase prior to further
purification. Since most antibiotics are sensitive to strong acids and bases, a weakly
[125]
acidic buffer solution has an advantage in this extraction . For example, a
Mcllvaine-EDTA solution was used to extract oxytetracycline in fish farm
sediment[126,127] . A citric acid buffer solution, in combination with ethyl acetate, was
[128]
used to extract tetracyclines in egg, poultry,fish and tissues This method was
also adapted to measure the concentration of tetracyclines and tylosin in fertilized
soil [129]. Both methods show a good recovery ratio for TCs, although weakly acidic
extractants are not suitable with macrolides and ionophore polyethers.

However, the use of phosphate buffer solution might cause degradation of the
[130]
analytes during the evaporation step, especially the TCs . Three different
antibiotic groups (TCs, SAs, and MLs) were simultaneously extracted in soil and pig
[131]
slurry using Mcllvaine buffer solution (pH 7.0) , with recoveries ranging from
27–51%, 68–85%, and 47–61% for oxytetracycline, sulfachloropyridazine and
tylosin, respectively, in a clay soil. However, the limit of detection was again too
high (18 µg/kg–40 µg/kg for soil and 70 µg/kg–140 µg/kg for pig slurry) to measure
most environmental samples. High performance liquid chromatography (HPLC)
equipped with mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is
the most widely used method to separate and quantify antibiotic samples. HPLC/MS
or MS/MS with electrospray ionization (ESI) has been used in several types of water
and food matrices [132, 133]

2 Grape Products

There are 16 bi-products made from grapes viz. raisin, grape juice, squash,
syrup, jam, jelly, vinegar, wine, pickles, chocolates, tartaric acid, oil, cattle feed,
tannin, etc. However, looking to the world scenario of different bi-products, it was
necessary to consider setting up of projects for manufacturing other value added
products from grapes.[134]. The Government of India has announced Agri Export
Zone (AEZ) for Nashik, Sangli, Pune, Solapur, Ahmednagar and Satara districts for

21
 Chapter 1

grapes and grape-wine parks in Maharashtra. The Government of Maharashtra had

nominated Maharashtra Industrial Development Corporation (MIDC) as a ‘nodal
agency’ for establishment of wine parks in Maharashtra state. Accordingly MIDC is
developing Godavari Wine Park at Vinchur, Nasik and Krishna Wine Park at Palus,
Sangli.

2.1 Raisin

Raisins are basically dry grapes and they are known as Kishmish, Bedana,
Manuka or dry fruit. During earlier days, grapes were dried on plants only which was
a very crude method resulting in wastages. Processing using some chemicals was
invented but there was considerable consumer resistance. Since then a new method
without use of chemicals, known as Australian method, has been introduced
successfully. Sangli and Nasik districts of Maharashtra grow large quantities of
grapes and many growers or gardeners are keen to supply to raisin makers due to
assured market. Maharashtra is, therefore, a preferred location. Raisin is a popular
dry fruit item with shelf life of around 6 months if stored properly. Apart from use as
a dry fruit item, it is used in large quantities in many sweet preparations, some farsan
items and desserts. Compliance under the FPO (fruit product order) and PFA
(prevention of food adultration) Act is mandatory.

Raisins are popular in India since long. Apart from regular use in many
preparations, a small quantity is also used in some herbal medicine preparations.
Grapes from Nasik and Sangli districts of Maharashtra are famous all over the
country. Grapes are perishable but raisins have a fairly long shelf life. There are
stockists at all major trading centres who buy large quantities as and when required
and then sell it to retailers in smaller lots. Normal demand is witnessed for raisins
throughout the year and it picks up during festive and marriage seasons. Demand
from individual households is not much but restaurants, star hotels, caterers and
sweet-meat makers are the major consumers. These two centres of Maharashtra
supply bulk quantities to western, central and north Indian states.

Raisins are made primarily by sun drying several different types of grapes.
They are small and sweetly flavored with a wrinkled texture. The technique for
making raisins has been known since ancient times and evidence of their production

22
 Chapter 1
[135-137]
has been found in the writings of ancient Egyptians . Currently, over 227
million kg of raisins are sold each year in the United States and the number is
expected to increased because raisins are recognized as a healthy snack. Most raisins
are small, dark, and wrinkled. They have a flavor similar to the grapes from which
they are made, but the drying process which creates them concentrates the amount of
sugar making them taste much sweeter. They are a naturally stable food and resist
spoilage due to their low moisture and low pH.

Raisins are composed of important food elements such as sugars, fruit acids,
and mineral salts. The sugars provide a good source for carbohydrates. Fruit acids
such as folic acid and pantothenic acid, which have been shown to promote growth,
are also significant components. Vitamin B6 is found in raisins and is an essential
part of human nutrition. Important minerals in raisins include calcium, magnesium,
and phosphorus. Additionally, iron, copper, zinc, and other nutrients are found in
trace amounts in raisins. Considering the composition of raisins and the fact that they
have no fat, it is no wonder that this fruit is considered a healthy snack.

The majority of grapes used for making raisins in the United States are grown
in California. This area has an ideal climate for grape growing because it has plenty
of sun during the summer and very mild winters. Five other countries, which
produce a substantial amount of raisins include Greece, Australia, Turkey, Iran, and
Afghanistan. Each of these countries have their own variety of raisin that they
consistently grow.

It has been evaluated in the European Union (EU) in the framework of

[138]
91/414/EEC Council Directive as a new molecule (Azoxystrobin) and was
approved in 1998 as an active substance of minimum purity 930 g/kg containing 25
[139]
g/kg Z isomer . It is a compound with no particular toxicological concerns, with
an acceptable daily intake (ADI) for man of 0.1 mg/kg b.w./day. On the basis of
critical good agricultural practice (GAP) in Europe, which refers to uses in Northern
countries, the value of 2 mg/kg was set as the EU maximum residue level (MRL) for
grapes [140]. According to the current EU legislation, in the case of dried or processed
products for which maximum levels are not explicitly fixed, the MRL applicable is
that for the fresh product taking into account, respectively, the concentration caused

23
 Chapter 1
[141]
by the drying process or the concentration or dilution caused by processing .
Trials carried out in Greece by the manufacturer on the Black Korinth variety of
grapes have shown that residues in raisins are higher than in the fresh commodity
(unpublished proprietary data).Procedures used in industrial food processing and
domestic cooking frequently have dramatic effects on residue levels [142-144]. In most
cases, large decreases in residues occur, as shown by Schattenberg et al.[145] in a
study to determine the effects of normal household preparation on pesticides in many
food commodities. However, residues may concentrate in processed commodities
following certain procedures. The ratio of the residue in the processed commodity to
that in the raw commodity is referred to as the transfer factor (TF). If this ratio is
greater than 1, the residue is said to concentrate upon processing. Studies on the
effect of processing and the determination of TFs have been recognized among the
[146]
main priorities in the process of MRL setting and for enabling a more realistic
[147]
exposure assessment and risk management . The aim of this work was to assess
the magnitude of residues of azoxystrobin on fresh grapes of the typical cv. Thomson
seedless (Sultana) and a seed-producing clone and on raisins produced from these
after the two types of processing commonly used, i.e., sun drying alone and sun
drying after treatment with a solution containing 3% K2CO3 and 1% ethyl oleate.

Azoxystrobin, a fungicide of the strobilurin group, has an European Union

maximum residue level (MRL) of 2 mg/kg for grapes. This work aimed to assess
residues on fresh and washed grapes and on raisins following processing with (i)
alkali treatment and sun drying and (ii) sun drying only. QUADRIS 25% SC was
applied according to good agricultural practice for two consecutive years on a typical
cv. Thomson seedless and a seed-producing clone. Samples were collected 0, 15, and
21 days post application and analyzed using gas chromatography/electron capture
detection; recoveries were 86 ( 12% for grapes and 99 ( 15% for raisins. Residues on
grapes were 0.49-1.84 mg/kg, and washing removed 75% of the residue. Residues in
raisins produced from seedless grapes were 0.51-1.49 (treatment 1) and 1.42-2.08
mg/kg (treatment 2), with residue transfer factors sometimes >1, even following
alkali treatment, which reduced residues considerably. To avoid trade problems, a
higher MRL for raisins is necessary [148].

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 Chapter 1

2.2 Wine

2.2.1 Wine Grape and Wine Production Scenario

The state of Maharashtra is undoubtedly the leader in both production and
consumption of wine in the country. It produces more than 75% of the total grape
and wine production in the country. At present Maharashtra totals 7,775 to 8,000
acres area under cultivation of wine grapes and it is increasing day by day to meet
the demand of new coming grape wine units. The total production of wine in India
was 22.5 million liters (from 62 established wineries). Out of this 21.1 million liters
wine is produced only in Maharashtra by 58 wineries. Nashik district, the wine hub
of India-Maharashtra, alone produced 9.4 million liters of wine in the year 2006 [149].
Gore and Sundar[150] estimated Indian wine consumption to be 1.1 million 9-liter
cases at a value of about US$ 60 million. With an annual growth rate of 20% to 25%,
consumption is projected increase to 2.0 million cases by 2011. On a per capita
basis, Indians consume only 9 ml wine annually as compared to 9000 ml in U.S. The
wine industry in India is in its nascent stages now; to follow quality parameters will
go a long way in making wines from India as a brand in International market.

2.2.2 Consumption and Market Analysis - Indian Wine Consumption

a. Tastes, Preferences and Presentation
The tastes and preferences of the Indian population err towards still wines
and more specifically, table wines. Though a market exists for champagne and
sparkling wines, these varieties sell at a much lesser rate (8-10% market share) 3. In
general, slightly sweet wines and the varieties of Sauvignon Blanc, Chenin Blanc,
Rieslings, and Gewürztraminer are fairly popular and also pair well with typical
Indian dishes. Similarly, rose and blush have been projected as good fits for the
Indian market. However, the majority of sales have stayed on traditional still red and
white wines. In regards to presentation, wine producers have two different
demographics in the Indian market upon which to focus: the upper class and the
general consumer. While the upper class prefers the classic presentation, i. e. real
cork, full bottle size, and dry red and white wines, the growing consumer class in
India gravitates towards approachable wine packaging, i.e. screw caps, half bottle
sizes, and sweet wines.

25
 Chapter 1

b. Value and Volume Estimates (2008)

For 2008, the authors of this study estimate Indian wine consumption to be
1.1 millon 9-liter4 cases at a value of approximately US$ 60 million5. With an
annual growth rate of 20% to 25%, consumption in this emerging market is projected
increase to 2.0 millon cases by 2011 (consumption projections, conservatively, are
4.0 millon cases by 2015 and 8.0 millon cases by 2020). On a per capita basis,
Indians consume about 9 milliters annually (compared to 9000 milliters in the U.S.)

c. Upper class Indians as Wine Consumers

This level of growth in the Indian wine market is in large part driven by the
upper class Indians, which is widely understood to be 2% of the population and
therefore approximately 20-25 million people. Many of these Indians have
increasing levels of disposable income and international experience and lifestyles
(either through studies, travel or work) that they have brought back to their country.
These changing tastes and preferences, coupled with higher levels of disposable
income and the increasing availability of domestic and imported wines, have resulted
in the emergence of India as a viable wine market.

d. Wine Consumption in Relation to that of Spirits and Beer

Indian alcohol consumption has traditionally focused on spirits and beer

instead of wine. Annually, Indians consume 50 millon cases of whiskey, 14 millon
cases of brandy, 25 millon cases of rum, 110 millon cases of beer, 200 millon cases
of country liquor, and 1 million cases of imported spirits (400,000 bottled imports
and 600,000 bulk imports which are bottled in India). This long-standing dominance
of spirits and beer as the alcohol beverages of choice among Indians has made it
difficult for wine to take a place in the market; however, despite this structure wine
is becoming more accepted, sought-after, and available.

e. Indian Wine Consumption by Location

The two largest and dominating markets in India are not regions, but rather
the city-areas of greater Mumbai and Delhi. It is estimated that as much as 65% of
total Indian wine consumption is accounted for in these two locations. This number
reaches an estimated 80% when including other major cities such as Bangalore,

26
 Chapter 1

Chennai, Kolkata (formerly Calcutta), Nashik, and Pune. This market dominance of
Mumbai and Delhi ensures their place as the fulcrum points for any producer or
distributor looking to increase sales to India. The city of Bangalore (with its high-
tech industry inflows), and the State of Goa (with its high energy tourism sector), are
a secondary, yet important focus for marketers as well. The cities of Kolkata,
Chandigarh, Nashik and Pune are all important niche markets and should be
followed and acted upon as appropriate. Chennai and Hyderabad have much
potential due to the growth of their IT industry but their government polies are not
yet conducive to wine sale.

f. Indian Wine Consumption - Historical and Projected (By volume)

The below figures for overall consumption are project through 2015. The
market share for imports remains steady for this analysis (at approximately 15%).
This market share, and its ability to increase, depends heavily on the level of
protectionism enforced by the federal and state governments in India. Should certain
onerous procedures and taxes be reduced (either voluntarily by India or through
dispute settlement proceedings in the World Trade Organization) the import market
share could rise significantly.

Table 1.5: Indian wine consumption (by volume)

YEAR TOTAL DOMESTIC IMPORTED

(Number of 9 Lit cases)
2004 550,000 470,000 80,000
2005 620,000 520,000 100,000
2006 750,000 630,000 120,000
2007 900,000 750,000 150,000
2008 1100000 920000 180000
2009 1400000 1180000 220000
2010 1700000 1440000 260000
2011 2,000,000 1700000 300000
2015 4,000,000 3,400,000 600,000
*Note: Figures exclude regional flavoured fruit wines. Figures are consumption
estimates

27
 Chapter 1

As elaborated on in the regulation section of this report, current government

measures to protect the nascent Indian wine industry have eliminated the ability of
imports to compete with domestic producers in a fair and open marketplace.

g. Wine Consumption for 2008 according to Price structure

Table 1.6: Wine consumption (by price structure)
DOMESTIC IMPORTED TOTAL
Under $10 600,000 - 600,000
Under $10 to under $20 250,000 50,000 300,000
Under $10 to under $30 60,000 90,000 150,000
$30+ 10,000 40,000 50,000
Total 920,000 180,000 1,100,000

The market share of imports for high value wines soar as cost increases into
the $20 + range. Domestic wines are still unable to demand a high price (during the
visit to India, the highest price domestic wine found by the authors of this report on a
menu was $23). Furthermore, the protectionist policies put in place at the federal and
state level in India have had their desired effect of ensuring that cheap foreign wines
cannot compete against domestics below $10/bottle.

h. Wine Consumption for 2008 by Wine type (Still Wines)

Traditional red and white varietal wines have gained a foothold in the emerging
Indian wine market. Rose wines have long been considered to be a good fit for the
Indian consumer, as they are thought by many to fit well with the Indian cuisine and
climate; however, they have consistently under-produced in the marketplace.
Table 1.7: wine consumption (by type)
Table Wines (9L cases)
Colour Domestic Imports Total
Red 480,000 120,000 600,000
White 420,000 50,000 470,000
Rose 20,000 10,000 30,000
Total 920,000 180,000 1,100,000

28
 Chapter 1

The dominance of red and white still wines in the marketplace reinforces the
assertion that the majority of wines sold in India are consumed by the upper 2% of
the population. Not surprisingly, this upper class has focused its consumption on
traditional wine types and styles.

i. Import Markets Shares by Country of Origin

Table 1.8: Import Market shares by country wise

Sr.No. Country Share by Country in %

1. France 45%
2. Australi 16%
3. Italy 11%
4. US 5%
5. S. Africa, Chile, New Zealand, Argentina 1-3.5
and Spain

The United States, while low at 5%, has only one way to go up. The
California lifestyle is appreciated in Indian culture. Additionally, while the majority
of wines are sold by France, the middle class of Indians have a palate that is more
accustomed to new-world style wines.

j. Indian importers (9L cases) top 107.

Table 1.9: List of importers in India.
Sr.No. Country 9L cases)
1. Brindco 51,000
2. Sonarys 24,000
3. Moet Hennessey 18,000
4. Global Tax Free Traders 14,000
5. Hema Connosieur Coll 13,000
6. Pernod Ricard 12,000
7. Sula 7,000
8. Fine Wines&More 6,500
9. Mohan Bros 4,500
10. T &G Trading 4,500
Note: The current number of wine importers in India is estimated to be 80.

29
 Chapter 1

k. Wine Consumption data for Imports

Table 1.10: Imports of Wine by India
India Import Statistics
Commodity: 2204, Wine Of Fresh Grapes, Incl Fortified, Grape Must O/T Heading 20.09
Year To Date: January œ November
Partner 2005 2006 2007
Unit USD Quantity USD Quantity USD Quantity
Country
World L 6775880 1252348 9304230 1563110 14413326 2833442
France L 3058019 472301 3939683 683768 6500437 867057
Australia L 794271 198358 1123938 236340 2315211 804071
Italy L 836427 138204 1275967 181825 1564735 254735
United States L 416491 62164 683059 93898 693722 92024
South Africa L 105043 46849 56070 105043 46849 56070
Chile L 288118 64447 250131 38086 335294 78997
New Zealand L 49367 7507 47574 5625 162757 27682
Argentina L 100126 27340 318751 35370 160324 32075
Spain L 159844 9994 84288 18284 151639 33389
Ref. Source: Global Trade Atlas: http://www.worldtradestatistics.com/gta

Those on this list, especially the top four, are well engrained in the existing market.

2.2.3 Pesticides Residues during Grape to Wine Processing

The main distribution area of the grapevine is in European countries. Among

these, France, Italy, and Spain control the sector on an international scale. Spain
dedicates the largest surface in the world to the cultivation of the grapevine (1230 x
103 ha, in 2001). The major part of this surface is inscribed in 56 Apellations d’
Origine Controlles (AOC). Three of these belong to Murcia (southeastern Spain), an
area of peculiar climatic characteristics that favor the development of pests and
diseases. The principal parasites of the vine are the grape moth (Lobesia botrana),
downy mildew (Plasmopora Viticola), powdery mildew (Uncinula necator), and, on
some occasions, gray mold (Botrytis cinerea) [151-154]. To control these parasites, vine
growers use insecticides and fungicides. This is important in maintaining grape
productivity and wine quality. However, in many cases, when the dose and/or the
established preharvest time for each product is not respected, hazardous residues are

30
 Chapter 1

left, and these become a permanent danger to the quality of the wine, the
environment, and consumer health [155-165]. In this sense, the elaboration method, the
correct winemaking processes, and the correct use of phytosanitary products are
influential in the dissipation and elimination of the current residues in grapes and
must. Most studies on pesticide residues deal with the transformation from vine to
wine, and the results reported show, on the one hand, their fate during vinification
and the influence of each technological process on the residue amount and, on the
other hand, that is almost impossible not to find residues in wine, even though at
very low or nondetectable levels [166-177].

The evolution of residual levels of four fungicides (cyprodinil, fludioxonil,

pyrimethanil, and quinoxyfen) during the elaboration of three types of wine with
maceration (traditional red wine, carbonic maceration red wine, and red wine of long
maceration and pre-fermentation at low temperature) and two types of wine without
maceration (rose´ and white) has been studied [178]. The disappearance curves of each
fungicide have been analyzed during the period of each winemaking process (21
days) and during the different enological steps involved in the elaborations. The
residual levels of fludioxonil reduce most quickly during the winemaking processes
without maceration, whereas the decrease in levels of pyrimethanil was the slowest
in practically all cases (with and without maceration). During carbonic maceration
winemaking, the decay constant of cyprodinil was greater than that of the other
pesticides in all assays (time and steps).

2.2.4 Heavy metals in Wine

Sodium arsenite is employed in viticulture as fungicide against the esca plant

disease (Eutypa lata). Arsenic is a suspected carcinogen and little is known about its
chronic sub-lethal effects. The concentration of As in wines mainly depends on
factors such as soil composition, grape variety, climatic conditions, use of pesticides,
process of vinification, and storage conditions. The Officer Internationale de la
Vigne et du Vin (O.I.V.) has set the maximum limit of As in wines at 200 pg/mL,
but in general it is accepted the presence of only a few pg/mL of As in
uncontaminated wines [179,-182] . Traditionally the determining of As in wine is based
on the generation of volatile amines followed by reaction with silver diethyl

31
 Chapter 1

dithiocarbamate, and measurement of molecular absorption of the As-containing

complex formed [183] . But the method LOD, estimated at 10 pg/mL, is not sufficient
for the wine analysis. Alternatively, hydride generation atomic absorption
spectrometry has been used for the quantification of several hydride-forming
elements in wine, including As[179].The technique normally requires sample
decomposition, a time consuming procedure, which may result in sample
contamination or analyte loss. Unfortunately, both spectral and non-spectral
interferences of As are present, in particular intensity of As signals are affected by
non-spectral interference of carbon containing substances. By ICP/MS signals of As
may be enhanced from carbon-containing compounds.

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