International journal of basic and applied research

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ISSN 2249-3352 (P) 2278-0505 (E)
Cosmos Impact Factor-5.86

Determination of residual aspects on surface after application of Imagard

Biquat disinfectant
Imran Memon1, Tahur Shaikh2, Idris Khan3 and Surjeet Samanta*
IMAGO & GETTER, Technical Team, Mumbai.

Abstract: The study was aimed to determine the residue of Disinfectant IMAGARD BIQUAT (Blend
of QAC and Biguanide), which is widely used for surface disinfection in Pharma manufacturing
industries, Institutional, Healthcare etc against harmful micro-organisms on equipment which can
come into contact with products. In some cases traces of the used compound(s) are inevitable.
Therefore, Maximum Residue Limits (MRLs) for the disinfectants involved are established by a fast
and simple analysis method to determine residue of disinfectant IMAGARD BIQUAT on test
surface sample was performed by high performance liquid chromatography - mass spectrometry
(HPLC-MS) and Photometric titration [15,16]. A convenient and reliable method for quantitative
determination of polyhexamethylene biguanide in aqueous solution has been developed, based
on complexation of the polymer with Naphthol Blue Black dye. Quantification of Quaternary
ammonium compound (QAC) and Biguanide on test surface is presented and discussed.
Keywords: Maximum Residue Limit (MRL), Disinfectant IMAGARD BIQUAT, High performance
liquid chromatography - mass spectrometry (HPLC-MS), Photometric titration.

Introduction
Recent developments include the use of disinfectants in industry as such, to reduce micro-
organisms [15]. An important group of combination in disinfectant formulations is the
―Quaternary ammonium compounds‖ often named ―QACs‖ and Polyhexamethylene Biguanide
(PHMB). Quaternary ammonium compounds especially those containing long alkyl chains and
PHMB is a cationic polyelectrolyte which exhibits broad antimicrobial spectrum. PHMB is well
soluble in water and has low biocide concentrations, causes minor foaming and does not leave
visible residues on surfaces of different materials [11]. Due to good germicide properties and low
toxicity PHMB is widely used as preservative in cosmetics, personal care products, antimicrobial
hand washes, wet wipes etc. Medical uses of PHMB include also prevention of microbial
contamination in wound irrigation and sterile dressings as well as disinfection of skin and
incisions [3,10]. IMAGARD BIQUAT disinfectant is a combination of Didecyldimethylammonium
chloride (DDAC) and Polyhexamethylene Biguanide (PHMB). The increasing use of disinfectant
across the pharma manufacturing industry, institution, healthcare etc. made necessary for this
testing and is critically discussed as some microorganisms may build up resistance phenomena
[4]. Considering this aspect, Limit of Quantification (LOQ) of QACs given by EU registration
discussion on surface, post application should be 10ppm [13]. As regular residue removal is
important for improved aesthetics and safety in cleanrooms. Therefore it was necessary to
develop a reliable method to quantify QAC and PHMB on surfaces.
Materials and methods
Disinfectant:
IMAGARD BIQUAT disinfectant product (Imago & Getter, Mumbai)

574 Received: 8 October Revised: 15 October Accepted: 20 October

Index in Cosmos
November 2018 Volume 8 Number 11
UGC Approved Journal
 International journal of basic and applied research
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ISSN 2249-3352 (P) 2278-0505 (E)
Cosmos Impact Factor-5.86

For Determination of DDAC:-

1. Preparation of Standard Sample:
15ml of Imagard Biquat Disinfectant sample was diluted to 1 Liter of Sterile Distilled water.
Transfer the sample to sterile tubes. Dilute further as per the requirement for HPLC-MS testing.
Calculate the concentration of Active content as given below in Table 1:

Preparation of Standard Dilution:

Table 1: IMAGARD BIQUAT disinfectant was diluted as 15ml in 1 litre of sterile distilled water.
Description Active Concentration (%)
Concentration of Actives (15ml in 1liter of Water) DDAC 0.150 %

Standard Dilution Prepared for HPLC-MS (10μl / 10ml) i.e. (10μl / 10000μl)

Concentration of Actives in Diluted sample DDAC 0.000150 %

(10μl/10ml)

2. Preparation of Residual Sample (Test Sample):

Dilution of (15ml in 1 Liter of water) Imagard Biquat Disinfectant sample was freshly
prepared in Sterile Double Distilled water. Sterile Stainless Steel surface/s {Dimension 15mm(L) x
15mm(b) X 0.5mm(h)} for surface disinfection was taken. Then dip the surface in freshly prepared
Imagard Biquat Solution and soak it for 15min. Remove the treated surface with the help of sterile
forcep and transfer it to sterile petri-dish. Air dry the surface in the Biosafety Cabinet for 15min.
After drying transfer the surface to fresh tubes containing Sterile Double Distilled water (20ml).
Shake the tubes for proper mixing and allow the tubes to stand for 15mins. Remove the test
surface from the tube. This tube will contain residual sample. Subject the sample as per the
requirement for HPLC-MS testing.

Apparatus:
An Agilent 1100 HPLC system (Agilent), consisting of a quaternary pump, a standard
autosampler and a column oven, combined with the mass spectrometer API 4000 (ABSCIEX), run
in ESI positive mode, was used for analysis of the sample. The sample was injected on a
Phenomenex Synergi Hydro RP 2.1 x 150 mm column with 4 μm particle size (Phenomenex).

For Determination of PHMB:-

Materials:
Polyhexamethylene biguanide hydrochloride [(C8H17N5HCl)m], has been obtained as 20 wt. %
solution of density 1.04 g/cm3. Stock solution containing 0.3 mmol/dm3 of PHMB mers has been
prepared and stored at 8 °C. The Naphthol Blue Black (NBB) dye has been purchased from Sigma-
Aldrich and was used without additional purification [14].
Apparatus:
Spectrophotometer Spectroquant Pharo 300 (Merck) using quartz cuvette with 1 cmoptical path.

575 Received: 8 October Revised: 15 October Accepted: 20 October

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November 2018 Volume 8 Number 11
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 International journal of basic and applied research
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Cosmos Impact Factor-5.86

Analytical Method

HPLC-MS/MS analysis (DDAC):

Mobile phase A consisted of 5 mM ammonium formate in purified water, mobile phase B
was 5 mM ammonium formate in methanol. A gradient program was used starting with a flow rate
of 0.1 mL per min and 100% of mobile phase A (0%B) at injection time, gradually changing to
30% A (70% B) over 3 min and to 15% A (85% B) until 6 min after injection. Then, the flow was set
to 0.3 mL per min and mobile phase composition was shifted to 10% A (90% B) over 3 min. This
mobile phase composition was kept for 11.5 min and was then shifted back to starting conditions
within 0.5 min. The conditions were maintained for 11 min for column equilibration. The injection
volume was 3μL, automatically diluted with 10μl of mobile phase A during injection procedure.
The column temperature was set to 40°C. ESI interface in positive mode was applied; the MS/MS
detection was performed in the multiple reaction monitoring (MRM) mode. The ion spray voltage
was 5500 V, the nebulizer gas and the turbo gas were synthetic air, both at 60 psi, and the curtain
gas was nitrogen at 30 psi, the source temperature was 500°C [16].
Two precursor-productions were monitored for each compound in the multiple reaction
monitoring mode (MRM). The first transition was used for quantification, while the second
transition served as a quality control [4].

Method validation (DDAC):

The method was validated at 0.1 mg kg-1 level for DDAC (n=5) following HPLC-MS/MS analysis [4].
All blank samples were proved not to contain QAC in relevant amounts right before validation.
We applied matrix matched calibration standards at concentrations representing 60 and 120% of
the validation level for each matrix and analyte. Quantification was carried out via calculating the
ratio between analyte peak area and ISTD peak area for both, calibration standards and recovery
samples. We then calculated the concentration of the recovery samples from the known
concentration of the calibration standard [5].

Photometric Titration (PHMB):

The photometric titrations have been performed measuring absorbance at 600 nm by means of
spectrophotometer Spekol 11 equipped with magnetically stirred 30 cm3 titration cuvette
with 2 cm optical path. The direct titration procedure has been performed as follows: 15 cm 3 of
PHMB solution of known concentration of mers in the range from 0.0533 to 0.1067 mmol/dm3
(obtained by mixing the stock PHMB solution with distilled water) has been titrated with 0.1 mM
NBB solution. The inverse titration has been performed as follows: 15 cm3 of 0.033mM NBB
solution has been titrated with PHMB solutions of known concentration of mers ranging from 0.1 to
0.3 mmol/dm3. Variance values were determined using tenfold repeating of the titration
procedures at the same initial conditions [14].

Results and discussion

Each compound could be detected in HPLCMS/MS with high sensitivity. The analytes
eluted as well defined and narrow shaped peak, for standard sample DDAC eluted at 12.454 min.

576 Received: 8 October Revised: 15 October Accepted: 20 October

Index in Cosmos
November 2018 Volume 8 Number 11
UGC Approved Journal
 International journal of basic and applied research
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ISSN 2249-3352 (P) 2278-0505 (E)
Cosmos Impact Factor-5.86

Table 2 gives an overview of the compounds peak, retention times, Area, Area% and Area sum
percent.
Also for residual sample, well defined and narrow shaped peak, DDAC eluted at 12.474
min. Table 3 gives an overview of the compounds peak, retention times, Area, Area% and Area
sum percent. Typical HPLC-MS/MS chromatograms of DDAC sample are shown in Fig. 1.

Fig 1. HPLC-MS/MS Chromatogram of (DDAC)

Table 2. Integration peak list

Peak Start Retension End Height Area Area% Area
time Sum
percent
1 1.019 1.085 1.302 990145 3745681 2.82 2.3
2 11.519 11.586 11.669 1141338 4304563 3.24 2.65
3 11.669 11.769 11.919 3037538 15381061 11.57 9.45
4 12.337 12.454 13.021 13562761 132979972 100 81.74
5 20.667 20.817 21.118 403026 4848396 3.65 2.98
6 26.359 26.593 27.144 81911 1431832 1.08 0.88
----------------------------------------------------------------------------------------------------------------

577 Received: 8 October Revised: 15 October Accepted: 20 October

Index in Cosmos
November 2018 Volume 8 Number 11
UGC Approved Journal
 International journal of basic and applied research
www.pragatipublication.com
ISSN 2249-3352 (P) 2278-0505 (E)
Cosmos Impact Factor-5.86

Results & Quantification:

Table 3. HPLC-MS Results of Standard and Residual sample
HPLC-MS Results Concentration (%)

DDAC

Percent of Active on HPLC-MS (Standard) at Retention time 81.74%

Percent of Active on HPLC-MS (Residual) at Retention time 73.16%

Quantification in Residual sample:

Conc.(%) of DDAC in Residual = Conc.(%) of Residual in HPLC-MS x Conc.(%) of DDAC in Std.
sample
Conc.(%) of DDAC in Standard sample (HPLC-MS)
= 73.16 x 0.000150
81.74

= 0.00013425 %
= 1.3425 µg/ml (ppm)

Direct Titration (PHMB) –

Titration of the polymer solution with the NBB dye solution (so called direct titration) gave
zigzag curves presented in Fig. 2. Location of the characteristic inflexion point on the obtained
curves, as well as the height of these bends, depends on the content of polymer in the analyzed
solution. Two methods of titration endpoint determination have been tested. The first one assumes
that the endpoint is the half-distance between the lines indicating the extremes of the titration
curve. The second method lets to determine the titration endpoint based on the second derivative
plot, which has been obtained from the polynomial function describing the titration curve [2,15].

Fig.2 Spectrophotometric direct titration curves obtained for different

PHMB mers concentrations (given in μmol/dm3) in the analyzed
solution using the dye solution with constant concentration

578 Received: 8 October Revised: 15 October Accepted: 20 October

Index in Cosmos
November 2018 Volume 8 Number 11
UGC Approved Journal
 International journal of basic and applied research
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ISSN 2249-3352 (P) 2278-0505 (E)
Cosmos Impact Factor-5.86

It turned out that the molar ratio of the NBB dye to the PHMB polymer at the titration endpoint was
1:2 leading to quite simple equation for PHMB concentration calculation:
C PHMB = 2 X C NBB X V NBB
V PHMB
where: CPHMB, CNBB — concentrations of the polymer mers and the dye, respectively, VPHMB,
VNBB—volumes of the polymer and the dye solutions, respectively.
As it can be seen in Fig. 3 the dependence between PHMB concentrations and titrant volume at
the endpoint has rectilinear character. Both the methods of endpoint determination provide very
similar results (Table 4). The calculated recovery values [CPHMB(determined) ·
100/CPHMB(prepared)] in most the cases fit the recommended range 95—105 %. Both the
half distance and the second derivative methods provide low values of variance, which are equal
to 3.5 x 10-6 and 5.9 x 10-6, respectively (at CPHMB = 0.0800 mmol/dm3). The limit of determination
for the direct titration procedure has been found equal to about 0.05 mmol/dm 3 of mers.

Table 4. Titrant volumes at the endpoint and recovery values for direct titration procedure
CPHMB V of CPHMB Recovery % V of titrant CPHMB Recovery
3
(prepared) titrant (determined) cm (determined) %
mmol/dm3 cm3 mmol/dm3 mmol/dm3
dalf-distance method second derivative method
0.0533 4.3 0.0573 107.5 4.1 0.0547 102.5

0.0667 5.0 0.0667 100 5.1 0.0680 102

0.0800 6.2 0.0827 103.3 6.3 0.0840 105

0.0933 7.1 0.0947 101.4 7.1 0.0947 101.4

0.1067 7.85 0.1047 98.1 7.9 0.01053 98.8

Discussion
Limits of Quantification (LOQ), defined as the lowest concentration showing a signal to noise
ratio >10, were well below 10ppm for all analytes. However, we detected small peak of QAC in
standard samples which was diluted at the lowest limit i.e. 10μl/ml as well as in residual sample
which was used without dilution. Although the LOQs were well below 10ppm in comparison to our
reporting limit which was set to 10ppm residue of QACs [8]. Small amounts of QAC seem to be
always present, which can be ascribed to the fact that QAC are commonly used in a broad
spectrum of cleaning agents and disinfectants and can also be present in indoor air or house dust
[7].
The recovery values in most the cases fit the recommended range 95—105 %. Low values of
variance, equal to 1.0 x 10-5 and 3.5 x 10-6 for the tangent intersection and second derivative
methods, respectively (calculated at CPHMB = 0.2 mmol/dm3) confirm good repeatability. The limit
of determination for the reverse titration procedure is about 0.1 mmol/dm 3 of mers.
579 Received: 8 October Revised: 15 October Accepted: 20 October
Index in Cosmos
November 2018 Volume 8 Number 11
UGC Approved Journal
 International journal of basic and applied research
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ISSN 2249-3352 (P) 2278-0505 (E)
Cosmos Impact Factor-5.86

Conclusion
We presented a simple and fast method to determine residues of quaternary ammonium
compounds (QAC) in residual samples. Quaternary ammonium compounds (QAC) show
antimicrobial activities and thus are used in disinfectant formulations. Therefore, they are widely
used in pharma manufacturing industry, food industry, healthcare etc. in order to clean surfaces
and ensure hygienic standards. A method applying the HPLC-MS technique was developed and
set up to provide reliable analytical results for samples to be tested. All compounds showed
satisfying recoveries while method validation. We set our reporting limit to 10ppm.
The anionic dye Naphthol Blue Black interacts with polyhexamethylene biguanide resulting in
reduction of solution absorbance at the molar ratio of NBB:PHMB mers equal to 1:2. Based on this
interaction, the new method of quantitative analysis of PHMB in water solutions has been
proposed, which consists in spectrophotometric titration at 600 nm wave length. Direct titration
procedure is applicable as it is more sensitive. The limit of determination (50 μmol/dm3 of mers)
is sufficient for polyhexamethylene biguanide assay. The present method was successfully
applied to the determination of trace amounts of PHMB on surfaces.
With rising health concerns, it became necessary to determine the residue of compounds
on the disinfected surfaces [5]. Residues of DDAC and PHMB have actually been found in a
residual sample subjected for testing but the results showed much lesser recoveries in
comparison to the set limit of 10ppm. The results exhibited exceptional reproducibility with more
than adequate sensitivity for monitoring down to current regulated levels. In addition, by using a
TOF detector, the combination of averaged MS spectra, mass accuracy checks and database
search results allowed for the definitive identification/confirmation of the QAC components [13].
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580 Received: 8 October Revised: 15 October Accepted: 20 October

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November 2018 Volume 8 Number 11
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581 Received: 8 October Revised: 15 October Accepted: 20 October

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