Direct Combination of Single Drop Microextraction with APMALDIMS

1
Direct Combination of Single Drop Microextraction
with AP-MALDI-MS
for
Rapid Screening of Low Molecular Drugs from Urine
samples
and
Quantitative Determination of Cationic
Surfactants from River and Municipal Waste Water
466.7

• Kamlesh Shrivas and Hui-Fen Wu
• Department of Chemistry,
• National Sun Yat-Sen University, Kaohsiung, 804,
  Taiwan
• Corresponding author. Phone [phone].
  
• Fax [phone].
• E-mail hwu_at_mail.nsysu.edu.tw.

368.5
284.5
304.3
2
Rapid Screening of Low Molecular Drugs from Urine
samples

• Present work reports the development of a new
  analytical procedure for simple and rapid
  screening of low molecular weight drugs (lt500
  Da.) from human urine sample by atmospheric
  pressure-matrix assisted laser desorption/ionizati
  on mass spectrometry (AP-MALDI/MS) combined with
  single drop micro extraction (SDME).

• The success of the proposed work was due to use
  of methyltrioctyl ammonium bromide (MTOAC) as
  additives to avoid signal to noise ratio of the
  matrix ions

• Also, SDME aided in the separation and
  enrichment of analytes from urine sample prior to
  AP-MALDI/MS analysis. This results in obtaining
  clean spectra and gain good sensitivity of the
  method

3

• The RSD and LOD of the method in urine sample
  were 8.2-14 and 0.3-1.6 µM respectively

• SDME was compared with liquid-liquid extraction
  (LLE) and hollow fiber-liquid phase
  microextraction technique to know compatibility
  of the present method in the extraction of drugs
  from urine sample.

• Thus, MTOAC as matrix ion signal suppressor and
  SDME as analyte separating devise in the
  AP-MALDI/MS analysis are shown to provide
  valuable information with simple, low cost and
  rapid screening of low molecular weight drugs
  from human urine sample.

4

• Matrix assisted laser desorption/ionisation mass
  spectrometry (MALDI/MS) is widely applied in the
  field of protein, oligosachharides, polymers and
  drug analysis. For higher molecular analysis of
  organic compounds by MALDI/MS is very successful
  may be due signal to noise ratio was negligible.

• However, analysis of low molecular compounds
  (lt500 Da) is still challenge for analytical
  chemist, because matrix noise signals usually
  complicate in interpenetration of sample signals
  in the lower molecular range

• The matrix organic compounds in use have low
  molecular lt 300 Da, due to this, they act as
  their own matrix during laser radiation and
  producing many matrix ions in mass spectra.

5

• In the present study, the background signal
  caused by the CHCA ions and matrices present in
  the sample was alleviated by the simultaneous use
  of two approaches.
• MTOAC as additive to reduce the matrix ion
  signal is the first and SDME was combined to
  separate and preconcentrate the drugs from human
  urine earlier to AP-MALDI/MS analysis being the
  second.
• The limit of detection and precision of the
  method in urine sample was investigated for the
  validity of the proposed method.

6
Diagram for SDME
7
INSTRUMENT The analyses of all drugs were
carried on Finnigan LCQ ion trap mass
spectrometer (Thermoquest Inc., San Jose, CA,
USA) equipped with an AP-MALDI/MS source. Mass
spectra were obtained in the positive ion mode
with laser power attenuated at 60 at 10 Hz.
Other operating conditions capillary temperature
250 oC, capillary voltage 40 V, tube lens offset
70 V and ion injection time 1070 ms,
respectively. The mass spectrometer was equipped
with nitrogen laser of 337 nm. All spectra were
scanning for 1 min in the mass range of 100-500
Da.
8
AP-MALDI/MS analysis of drugs assisted with SDME

• CHCA is widely used as matrix in the MALDI-MS
  analysis for peptides and drug due to its
  homogenous crystal formation with analytes. Also,
  it is good absorber and transfer of laser energy
  to analytes through ionization process

• But actual problem was associated during of
  analysis of lower mass organic compounds (lt500
  Da.).

Figure 1 shows AP-MALDI-MS spectra of all drugs,
nortriptyline (NT), amitriptyline (AT),
imipramine (IP), trimeprazine (TM) and quinine
(QN) at m/z 264.5 (NTH), 278.1 (ATH), 281.2
(IPH), and 325.4 (QNH), prior to SDME at CHCA
concentration of 0.028 M (without MTOAC).
9
ATH
 
278.1
Abundance
QNH
NTH
325.4
IPH
264.5
2 CHCA-CO2H
2 CHCAH
TM
378.9
281.3
CHCA-H2OH
CHCAH
298.2
190.4
335.4
172.5.
m/z
Figure 1. AP-MALDI/MS spectra of spiked drugs (11
µM) from deionized water using SDME at CHCA
concentration of 0.026 M.
10

• Figure 1, also represents the AP-MALDI/MS
  spectra of matrix ions observed at m/z 172.5
  (CHCA-H2OH), 190.4 (CHCAH), 335.4
  (2CHCA-CO2H) and 378.9 (2CHCAH).

• These are the prominent peaks, which could
  create confusion in the identification of analyts
  in the low-mass range analysis. These spectra
  show the domination of the matrix signal over
  analyte signals.

• In order to suppress the signals of matrix ions
  and to obtain clean spectra of the drugs of
  interest, MTOAC was added

• Figures 2 (a) to 2 (c) show the AP-MALDI/MS
  spectra of drugs after applying different molar
  concentration ratio of CHCA/ MTOAC from 100001
  to 7001, prior to separation of drugs by the
  SDME.

11
368.5
700000
350000
379.1
278.1
281.1
298.1
325.5
172.1
264.3
190.3
100000
300
500
150
m/z
Abundance
368.5
120000
600000
278.1
379.1
281.1
325.5
264.3
298.1
172.1
190.3
100000
300
150
500
m/z
368.5
120000
600000
278.1
281.1
325.5
264.3
298.1
100000
500
150
300
m/z
12

• Figure 2 (a) displays the intense peak of
  surfactant produced at m/z 368.5 (MTAO) by
  losing Cl- ion from MTOAC.

• It was observed that the proper fixing of
  CHCA/MTOAC concentration ratio in AP-MALDI/MS
  analysis of drugs before to SDME was very
  significant.

• The use of higher concentration of MTOAC may
  cause suppression of drug signal peak (not shown)
  and lower concentration could cause more signal
  to noise ratio by the matrix (CHCA) as shown in
  figure 2 (a) and 2 (b).

• The optimum ratio of CHCA/MTOAC for obtaining
  clean spectra for the determination of drugs
  prior to SDME was 7001, shown in figure 2 (c).
  Hence, this optimum ratio of CHCA/MTOAC was used
  for further investigations.

13
Optimization of Single Drop Microextraction
Extraction efficiency of each drug was observed
by the abundance of signal of three replicate
analyses. The results obtained for optimization
of extraction parameters are given in table 1.
Optimum extraction efficiency of drugs were
obtained at the following conditions xylene as
extracting solvent, stirring speed 240 rpm,
extraction time 10 min, exposure volume of
acceptor phase 1.0 µL and salt addition of 10
at room temperature.
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15
APPLICATION

• The precision of the proposed method was checked
  by replicate analysis (n6) of 3.5 µM of drugs
  in urine sample at the optimized condition of
  SDME. The RSD of the method was lt 12.

The LOD values obtained for drugs in urine were
found from 0.3-1.6 and 11-18 µM with and without
use of MTOAC respectively are given in table 2.
10-43 folds of improvements of LOD were obtained
when the MTOAC was used as additive to suppress
the peaks of matrices ions.

• This results showing that SDME can also useful
  excluding interference caused by the matrices in
  the urine samples. Also, 13-52 folds enrichment
  factors were calculated at concentration of 1.6
  µM were spiked in the urine samples

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17

• Comparision of microextraction techniques
• The LOD values for the SDME, HF-LPME and LLE
  were 0.3-1.6, 0.3-2.3 and 11-23 µM, respectively.

• Compared to the customary LLE method, the
  current SDME offers better sensitivity.
• The limit of detection acquired by the present
  method was as good as the HF-LPME.
• However, SDME was recognized as simple and
  faster than HF-LPME may be due to the direct
  suspending of organic solvent in the tip of
  syringe for extraction of analytes from aqueous
  phase, instead of placing solvent in to hollow
  fiber (extra steps were involved cleaning of
  hollow fiber before placing solvent).

18

• CONCLUSION
• This method is proved to be simple and rapid
  method for screening of low molecular drugs from
  urine sample.
• The background signal to noise ratio is very
  negligible when MTOAC used as additives.
• Separation and preconcentration excludes most of
  the interference during the analysis of drugs
  from urine sample by SDME. Both the approach
  helped in reducing matrix interferences.
• This method could act as model for monitoring
  low molecular drugs in biological samples, urine
  and plasma.

19
Quantitative Determination of Cationic
Surfactants from River and Municipal Waste Water
20
Summary of the proposed work

• Method was based on the extraction of CS from
  aqueous sample into 2 µL of organic solvent by
  single drop micro extraction (SDME).
• Solvent Octanol
• Stirring speed 200 rpm
• Extraction time 7 min
• Acceptor phase volume 0.8 µL
• No salt addition
• At room temperature
• Then, it was analyzed by AP-MALDI-MS

21

• The enrichment factor for CS was found to be
  40-64 folds for 7 min of extraction at room
  temperature. The linearity and limit of detection
  (LOD) of the method were 50-1500 and 10 µg/L,
  respectively.
• The relative recoveries in river and municipal
  waste water were found to be 95.2 to 105 and
  90.0 to 100 respectively and this shows good
  reliability of the method.
• The enrichment and LOD values obtained by SDME
  were compared with LLE and HF-LPME, and found
  SDME is compatible with HF-LPME and much better
  than traditional LLE.

22
INTRODUCTION
Cationic surfactants (CS) are surface-active
compounds with at least one hydrophobic alkyl
chain and a hydrophilic group carrying a positive
charge. CS are positively charged in aqueous
solutions. A positively charged quaternary
nitrogen atom characterizes the quaternary
ammonium compounds.
Hydrophobic (water repelling)
Hydrophilic (water loving)
CH3
N
H3C
CH3
CH3
Head
Tail
23

• CS are used
• Hair rinsers Textile softener
• Preservatives or antiseptic agents in industrial
  and commercial products.
• Due to their ability to stabilize emulsions and
  their antibacterial properties, surfactants used
  widely in cosmetics, drugs and microbiocides.
• CS are also widely used in the manufacture of
  commodity samples, i.e. detergents, soaps,
  shampoo, etc. as surface cleaning agents

24
Toxicities of CS

• CS are toxic to aquatic invertebrates as
  indicated by study of Belanger et. al.
• The few available absorption studies conducted
  with CS indicate that absorption occurs in small
  amounts through the skin.
• CS also can causes, i.e. nausea, vomiting,
  diarrhea, dermal necrosis, lung complications,
  hypotension, corneal damage.

25

• The most common method to determine CS
• Spectrophotometry
• High performance liquid chromatography (HPLC)
• Electrospray ionization mass spectrometry
  (ESI-MS)
• Capillary electrophoresis (CE)
• Ion pair chromatography (IC)

26
Sample preconcentration and extraction of analytes
Liquid-liquid extraction (LLE) is one of the old
conventional methods used for extraction of
analytes from various matrices.
Drawbacks more solvent consumption, tedious,
time consuming and exposure of toxic chemicals
during operation.
27

• Application of Matrix-assisted laser desorption
  /ionization-mass spectrometry
• Powerful sensitive technique Proteins, nucleic
  acid, drugs and synthetic polymers.

• Major advantages of the technique simplicity of
  usage, high sensitivity and high sample
  throughput.

• Quantitative analysis of different analytes
  using internal standard.

• The properties of internal standard should be
  chemically similar to the analyte, completely
  resolved from the analyte peak and internal
  standard should be near to the sample of analyte.

28
Atmospheric pressure-matrix assisted laser
desorption/ionization mass spectrometry
(AP-MALDI/MS) All mass spectra were collected in
positive ion mode by using an AP-MALDI ion source
(Mass Tech Inc., Columbia, MD, USA) in
conjunction with an ion trap mass spectrometer
(Thermo Finnigan LCQ-Advantage, San Jose, CA,
USA). Analytes were irradiated with a nitrogen
laser at 337 nm. Bombarding of laser beam on
target plate was fixed for 1 min at 1.8 kV for
all spectra and mass range was set at 100-600 Da.
The laser power was operated at 10 Hz. The
voltage of capillary and tube lens offset was 40
V and 70 V, respectively. The temperature of
capillary was constant at 250 oC. Mass spectra
were obtained by summing all individual shots.
29
RESULTS AND DISCUSSION
Simultaneously quantitative analysis of cationic
surfactant mixtures by AP-MALDI mass spectrometry

• MALDI/MS is difficult in the analysis for low
  molecular weight compounds (MW lt 500 Da). This is
  due to the interference of matrix ions.

• In this study, the determination of mixtures of
  CS including CTAB, CPC and MTOAC from water
  samples was easily performed since the matrix
  ions of CHCA were suppressed in the presence of
  the surfactants.

30
M-Br- of TOAB (IS)
466.7
Figure 8
M-Cl- of MTOAC
Abundance
368.5
M-Cl- of CPC
M-Br- of CTAB
284.5
304.3
m/z
Figure 4 presents the SDME/AP-MALDI mass spectra
of CS. The ions shown at m/z 284.5, 304.3,
368.5 and 466.7 were assigned as M-Br- of
CTAB, M-Cl-of CPC, M-Cl- of MTOAC and
M-Br-of TOAB, respectively. These ions were
produced by elimination of a halogen ion (Br -,
Cl -) from the molecular ion.
31

•  
• Optimization of extraction parameters for
  single drop micro extraction (SDME)
• Efficient extraction of CS into organic
  acceptor phase (organic solvent) was obtained by
  optimizing sampling conditions.
• Extraction solvent, stirring speed of sample,
  extraction time, acceptor phase volume and salt
  addition.
• The extraction efficiency was based on the
  abundance of each analyte for three replicate
  analyses.

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Solvents



 

 
 
33
Stirring speed
34
Extraction time
35
Acceptor phase
36
Salt concentration
37
Enrichment factors, calibration curve and
precision

• Enrichment factor is ratio of concentration
  between organic solvent and aqueous phase used
  for extraction. Enrichment factors were
  determined to know extraction efficiency of CS
  at optimal conditions.

• Signal intensity of calibration and internal
  standards were recorded at positive ion mode in
  AP-MALDI-MS. The ratio of signal intensity of
  analyte to internal standard were calculated and
  used to draw calibration curve.

38

• The LOD were calculated as the amount of
  compound that would still give signal three times
  than the noise. The LOD for all surfactants were
  found to be 10 µg /L.

• For quantitative AP-MALDI/MS analysis of CS, an
  internal standard was required to improve
  reproducibility of the results.

• The precision of the method was expressed as the
  relative standard deviation (RSD) of replicate
  analyses (n6), at concentration of 1 mg/L
  carried out within working day.

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40
Application
41

• Relative recoveries were estimated as intensity
  ratio of CS in matrix sample to the intensity in
  deionised water spiked of same amount of
  analytes.

42

• Table 4 displays the comparison of LODs and
  enrichment factors of SDME with HF-LPME coupling
  to AP-MALDI/MS for cationic surfactants in
  deionized water. The LODs for SDME and HF-LPME
  were 10 and 5-8 µg/L, respectively.
• The LOD values obtained by SDME were compatible
  with the HF-LPME. The enrichment factors obtained
  by HF-LPME were 48 to 62 folds, which were
  comparable to SDME.
• However, the SDME is more convenient and lower
  cost than the HF-LPME since no hollow fiber is
  required in the extraction processes.

43

• CONCLUSION
• SDME combined with AP-MALDI-MS
• proved to be a simple, rapid, and selective
  method for the simultaneous quantitative
  determination of cationic surfactants from river
  and municipal waste water.
• Large number of water samples could be screened
  in short period of time to know exposure of these
  cationic surfactants in the environment.
• This effort could be certainty helpful for
  quality control of pollution in different
  compartments of the environment.