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Devise a rapid and non-destructive mass spectrometric method to detect and quantify
the presence of Risperidone and its major metabolite 9-Hydroxyrisperidone in a
fingertip smear.
By Molly Winterbo.om
Table of Contents
Abstract.............................................................................................................................1
Characteris*cs of Risperidone.....................................................................................................2
Previous techniques used ...........................................................................................................3
Prior to running the method .......................................................................................................4
Method..............................................................................................................................5
Instrumenta*on Requirements/sugges*ons: ..............................................................................5
DESI ...............................................................................................................................................................5
DMS...............................................................................................................................................................6
MRM..............................................................................................................................................................7
MS-TOF..........................................................................................................................................................7
Quan*fica*on of Results.............................................................................................................8
Alterna*ve future adapta*ons to the method.............................................................................9
Conclusion .........................................................................................................................9
References .........................................................................................................................9
Abstract
The aim of this paper is to iden0fy and possibly develop an analy0cal chemistry approach to
detect and quan0fy the presence of an an0psycho0c drug known as Risperidone and its
major metabolite 9-Hydroxyrisperidone, present in a finger0p smear during a rou0ne Drug
monitoring check. Usually, these checks would be carried out on the pa0ents’ blood plasma
or urine sample. However, this paper gives way to a new route of carrying out this check,
using a finger0p smear sample given by the pa0ents, which will provide an alterna0ve, faster
and less invasive sample collec0on, which is also less likely to be tampered with by the
pa0ents. This method combines the use of DESI (Desorp0on electrospray ionisa0on), DMS
(Differen0al Mobility Spectrometry), MRM (Mul0ple-reac0on monitoring) where the sample
will enter a triple quadrupole, this is then coupled with a MS-TOF (Time-of-Flight) tandem
MS. Using this method will enable a non-destruc0ve analysis of the finger0p smear, with a
possibility of detec0ng the enan0omers of Risperidone and it’s metabolite 9-
Hydroxyrisperidone for further future analysis if required. Here, the details of each
component and its benefit will be explained along with any alterna0ve method that could be
used and any previous methods that have already been used when detec0ng the drug in
blood plasma or urine.
Characteris*cs of Risperidone
Figure 1. Molecular structure of Risperidone
As Risperidone is metabolised in the body it results in the forma0on of 2 enan0omers due to
its chiral centre, (+)-9-Hydroxyrisperidone and (-)-9-Hydroxyrisperidone1
. Chirality plays a
significant role in the detec0on and quan0fica0on of drug substances, however in the case
of Risperidone the enan0omers are similar in their effects when administered to pa0ents2
so
the desire to separate the metabolites using specific methods is not needed but this
technique could be used for this purpose.
Figure 2. Molecular Structure of 9-Hydroxylrisperidone, the metabolite of Risperidone
Based off previous studies we can determine Risperidone’s m/z in a range of 411 to 191, its
metabolite 9-Hydroxyrisperidone in the range of 427 to 2073
, therefore these will be the
values set at the quadrupole in the MRM phase of the instrument to find the m/z peak and
the peak of its product ion in order to help the quan0fica0on process at the end of the
analysis.
Due to the presence of heterocycles containing nitrogen and a piperidine ring, this indicates
that the basic proper0es present in Risperidone makes it suitable for protona0on using DESI,
more specifically the two benzisoxazole rings that contain nitrogen atoms will be protonated
in a posi0ve ionisa0on mode4
. From previous studies on Risperidone and its analysis using
mass spectroscopy also indicated that it would ionise under posi0ve ion condi0ons,
producing the [M+H]+
ions.5
Figure 3. Fragment ions that will produce a high intensity peak on the m/z spectra, Risperidone fragment ion on the Right
and 9-Hydroxyrisperidone fragment ion on the LeC.
Other fragment ions can be seen in the spectra, see figure 7 for the spectra along with the
fragment ions or Figure 3 for the fragment ions. These fragments contain the change
between Risperidone and 9-Hydroxyrisperidone due to the presence of an OH group a^er it
has been metabolised.
From looking at Risperidone and its metabolites structure, it presents polar characteris0cs
due to it containing nitrogen, promo0ng electronega0vity in the compound, especially being
encompassed in the heterocyclic aroma0c rings. So again, the use of posi0ve ionisa0on will
occur.
Previous techniques used
The monitoring of Risperidone adherence in the body has been recorded previously in many
studies using various analy0cal techniques including LC-MS/MS or HPLC/ MS/MS as the
technique of choice, due to its fast, sensi0ve, and selec0ve analysis. However, these benefits
can also be its downfall when it comes to the analysis of drugs such as Risperidone and its
metabolite 9-hydroxyrisperidone. It’s fast paced analysis increases the probability of the
sample of interest elu0ng from the column with another interfering compound mixed within
the sample taken from pa0ents urine or blood plasma6
, or in this case fingerprint smear. In
this paper, the aim is to devise a technique to be used to determine the detec0on and
quan0fica0on of Risperidone and 9-Hydroxyrespirdone in a fingerprint smear given from
pa0ents during a drug adherence examina0on without the use of HPLC to avoid the
possibility of the analyte elu0ng from the column undetected.
Many of these techniques paired their HPLC with UV detectors, which is common prac0se
due to its ease of use and reliability. It’s benefits include the fact that it is a non-destruc0ve
technique and that it can provide informa0on on peak iden0ty. However, in order to detect
using UV the analyte must have chromophoric ac0vity7
, which is the presence of a
chromophore group in the molecule that has the ability to absorb light energy in the visible
spectrum8
. Risperidone is a non-chromophoric an0psycho0c compound, so UV-detec0on
would not be suitable to quan0fy the presence in the finger0p smear of the pa0ents.
Prior to running the method
Before running any actual finger0p smear, the first thing would be to calibrate the machine
using standards of Risperidone and its metabolite. This should be done in order to produce a
calibra0on curve to enable effec0ve quan0fica0on of the finger0p smear at the end of the
test. Standards such as Risperidone tablets can be used in varying doses such as 1,2 and 3mg
tablets9
, this can then be used to create standard solu0ons with varying concentra0ons by
dissolving and dilu0ng in solvents such as methanol, enabling droplets to be placed onto a
glass microscope slide and run on the machine un0l the instrument starts to pick up and
detect the standards, producing its peaks. Alongside the analysis of the standards the
solvent, mobile and sta0onary phases would be altered to increase the peak detec0on.
Figure 4. Example of a Standard calibraFon curve of Risperidone10
A^er a standard calibra0on curve has been produced (see Figure 4 for an example of a
calibra0on curve), and reproduced for method valida0on, a sample of a finger0p smear can
be taken from a pa0ent, disregarding which finger but making sure to take at least 3 per
pa0ent to be able to provide reproducibility and run mul0ple fingerprints for the detec0on
of Risperidone per pa0ent. It may also be beneficial to overlap all 3 finger0p smears on 1
slide to make sure enough of Risperidone is present in order for it to be detectable and
quan0fiable.
Another step to consider is sample prepara0on prior to placing on the DESI sample stage. A
suggested technique to collec0ng the finger0p smears would be to have the pa0ents clean
their hands in 100% ethanol solu0on in order to prevent further contaminants from being
placed onto the glass slide with the finger0p smear11
. This step will help reduce peaks of
non-interest to the chemist and help the selec0vity for trying to detect Risperidone and 9-
Hydroxyrisperidone.
Method
Scheme 1. Flow chart of the suggested method
Shown above is the layout of the suggested method that could poten0ally be used to
analyse the presence of Risperidone and 9-hydroxyrisperidone in pa0ents finger0p smear.
This paper will now discuss each component of this method in further detail along with
explana0ons as to why this specific system has been chosen.
Instrumenta*on Requirements/sugges*ons:
The solvent suggested to use would be a ra0o of 95:5 v/v methanol: water with 1-5% ace0c
acid modifier. However, if the user would like to op0mise the solvent use themselves, it
would be done by star0ng off with a 50:50 v/v methanol: water working their way up to the
95:5 v/v along with adjus0ng the ace0c acid modifier from 0.1% up to the suggested 5% to
see which works best and gives a peak with the best shape and intensity.12
The spray voltage can be monitored by varying its voltage range from around 4 to 6kV, this
will promote efficient ion genera0on. The spray solvent flow will be measured in microL/min
increments balancing out stability13
. Within the DESI source, the probe height an angle
would need altering to op0mum coverage of the fingerprint smear, this would be done by
adjus0ng the distance from the surface from around 1mm to 4mm and an angle from
around 40 to 60 degrees.14
DESI
Using a more non-destruc0ve method to start the analysis process like DESI, rather than
MALDI, would allow liile upfront sample prepara0on which would also allow less tampering
with the finger0p smear.
Figure 5. Diagram of DESI system.15
By taking the finger0p smear directly onto a glass microscope slide and placing it onto the
automated moving stage under a DESI probe (See Figure 5), the DESI would be able to apply
the charged solvent droplets (see instrumenta0on Requirements/sugges0ons for more
informa0on) from the probe onto the sample, making sure to cover the en0re fingerprint
smear, including any standards doied on the smear or around it on the edge of the glass
slide, if the standard calibra0on is being run alongside the fingerprint smear analysis. The
finger0p smear sample could be washed with water in a squeeze boile prior to running on
DESI, to remove any compounds that could interfere with the detec0on of Risperidone, but
if the pa0ents had washed their hands with the 100% alcohol first then it wouldn’t make
much difference. Using water will not affect Risperidone’s detec0on as it has poor aqueous
solubility.
A key step to take when using DESI for ionisa0on is making sure to have con0nuous and
op0mal scan of the fingerprint smear. In order to do this, the ion intensi0es of the Peak of
interest (Risperidone, 9-hydroxyrisperidone and the standard) must be monitored by the
adjustment of the electrospray incidence angle, the distance between the sample surface,
emiier, and MS inlet capillary, also the distance between the sample surface and the ion
collec0on capillary16
.
DMS
A^er the sample has been ionised, the ions will be carried away by Nitrogen, the carrier gas
in DMS where they will be separated based on differences in ion mobility in an electric field,
adding another dimension of separa0on to the analy0cal technique as the results will
include Risperidone and its metabolite, so the more separa0on will be needed to avoid any
peak overlaps. A separa0on voltage will force the ions to move closer to one electrode than
the other planar electrode. To stop the ion of interest going to the electrode a counter
voltage (compensa0on voltage or direct current voltage) is applied to the system17
. These
voltages need to be altered and tweaked to only enable the ions of interest through to the
first quadrupole in MRM.
Figure 6. Image of how DMS ion mobility works.18
Since the sample will include Risperidone and its metabolite, there will be different mobility
profiles, therefore using DMS coupled to DESI would improve selec0vity of detec0on. Along
with the removal of any background noise in the spectra and providing cleaner peaks for
further analysis. It will also provide rapid separa0on of Risperidone’s isomers if further
analysis is wanted on the specific isomer men0oned in characteris0cs of Risperidone sec0on
of this paper.
MRM
The use of MRM on a triple quadrupole mass spectrometer would allow the operator to
select the target m/z for both Risperidone and 9-Hydroxyrisperidone allowing those ions
with that mass to charge ra0o through to then be fragmented at the second quadrupole and
from there a specific fragment can be selected at the third quadrupole.
Based off of previous studies, the first quadrupole will be set to the condi0ons where
Risperidone’s m/z in a range of 411 to 191, its metabolite 9-Hydroxyrisperidone in the range
of 427 to 207, allowing only these ions through to be fragmented at the second quadrupole
producing the fragment ions [C23H27FN4O2]+
for Risperidone and [C23H27FN4O3]+
for 9-
hydroxyrisperidone, to which the third quadrupole would be set to only let these
fragmented ions through to the TOF.
Using MRM would be profoundly beneficial as it would provide highly sensi0ve and selec0ve
quan0fica0on of the analyte of interest (Risperidone and its metabolite) whilst using it
alongside internal standards that would be recognised by the database19
. This highly
sensi0ve and selec0ve system will enable the analyte to be picks on its m/z value, therefore
monitoring specific precursor and product ion transi0ons, resul0ng in a significant decrease
of background noise and interferences20
.
Although using MRM can be a costly method of choice, it may be the only suitable op0on to
use due to its ability to carry out target analysis on the compound of interest. This con is
then outweighed by the benefit of the system and the use it is intended for.
MS-TOF
Aiached to a single quadrupole for the ions to simple pass through, the high mass accuracy
and resolu0on of TOF (Time-Of Flight) will enable accurate mass measurement of the
precursor and fragment ions for each compound detected by MRM, allowing the
confirma0on of the molecular formulas for both Risperidone and its metabolite. As a TOF
mass analyser works by separa0ng the ions based on their velocity the assump0on is that
the ions with the smaller m/z will move more quickly through the chamber of the TOF and
be detected first21
. In this case, based on the informa0on stated, another assump0on can be
made sugges0ng that the ion for Risperidone will hit the detector first as it has the smaller
m/z value for the precursor and fragmented ions compared to the ions for 9-
hydroxyrisperidone.
Finishing the method by adding a MS-TOF will further increase the instruments sensi0vity in
being able to detect the analytes of interest, along with its resolu0on and mass accuracy.22
However, using an MS-TOF has been known to have some limita0ons around ionisa0on
source interac0ons, mass accuracy dri^ and data processing. So, during the trial, checks may
have to be carried out to make sure the TOF analysers don’t dri^, this can be done with the
use of internal standards. Looking at the data processing aspect, that is subjec0ve to the
analyte in ques0on and what the user wants to analyse. The immense full spectral data
produced by the MS-TOF can be overwhelming to the computer so^ware23
and the user, so
it may be beneficial to look at an alterna0ve to MS-TOF like a triple quadrupole, Orbitrap or
Ion Trap Mass Spectrometers.
Quan*fica*on of Results
From looking an example spectrum taken from a paper, Risperidone and 9-
hydroxyrisperidone can be detected by finding its m/z peak and the product ion peak.
Looking below at Figure 7 the spectra show Risperidone and its metabolite being detected at
the values set at the triple quadrupole during MRM.
Figure 7. Example spectra of Risperidone and 9-Hydroxyrisperidone24
By using the calibra0on curve produced from the standards of Risperidone, the
concentra0on of the sample can be measured and quan0fied by finding the area of the peak
produced in the sample MS spectrum and using a line of best fit along the graph and looking
at the x-axis for the concentra0on value. See Figure 4 for an example of a calibra0on curve
produced from a Risperidone standard.
Since Risperidone and its metabolite aren’t a protein, it would not work to perform a de
novo analysis on the spectra, so the best way to quan0fy the results would be to compare
using a calibra0on curve that was produced on the same machine using a standard of
risperidone and possibly making another curve for a standard of its metabolite to use that
for quan0fica0on on 9-hydroxyrisperidone.
Alterna*ve future adapta*ons to the method
Stated in the DESI sec0on of this paper an alterna0ve to using this ionisa0on technique
would be to use MALDI (Matrix-Assisted Laser Desorp0on/ionisa0on), although this method
would be more destruc0ve on the sample as it uses a UV laser to transfer energy to the
matrix. Another alterna0ve in this instrumenta0on method would be the use of DIA (Data-
independent Acquisi0on) coupled with Dri^ mobility in place of MRM and DMS, although
Dri^ mobility would have to occur a^er DIA and the quadrupole in order to separate the
parent and daughter ion peaks in the spectra.
As discussed previously in the MS-TOF sec0on of this paper, an alterna0ve to use for the
detec0on of the precursor and fragment ions would be a triple quadrupole, Ion trap or
Orbitrap Mass Spectrometers. The use of a Triple Quadrupole would enable superior
quan0ta0ve accuracy and precision when targe0ng specific analytes, however, with this
proposed technique the incorpora0on of a triple quadrupole has already been installed with
the use of MRM25
. Another alterna0ve could be the use of an Orbitrap. The Orbitrap could
be considered very similar to the MS-TOF analyser as it also offers high mass accuracy and
resolu0on26
.
Conclusion
In conclusion, the development of the method incorpora0ng the components of DESI-DMS-
MRM-MS-TOF has been produced based off of previous studies done on the detec0on and
quan0fica0on of Risperidone and its metabolite 9-hydroxyrisperidone in pa0ent’s blood
plasma or urine or the comparison of different tablets of Risperidone produced by various
manufacturers in different countries, the main focus on those papers being the
standardisa0ons techniques used. Although in theory this method will work in producing
detectable and quan0fiable ions of the analyte of interest, the only way to jus0fy and
thoroughly validate this method would be trialling it in a lab under the right condi0ons, with
suitable and willing pa0ents, carrying out mul0ple robust tes0ng with a wide range of
finger0p smears.
References
1 Michael G. Aman, Et al, Clin Ther. 2007 Jul, 29, 1476-1486
2 M.C. Mauri, S. PaleVa, M.Maffini, Excli Journal, 2014, 13, 1163-1191
3 D E. Moody, J D. Laycock, W. Huang, and R L. Foltz, Journal of Analy6cal Toxicology, Vol 28, September 2004
4 R.G. Cooks, Z. Ouyang, Z. Takats and J.M. Wiseman, Ambient Mass Spectrometry, Science, 2006, Volume 311, Pages 1566-
1570.
5 A.E. Steuer, A.B. Poetzch, M.R.Baumgartner, S.D Kraemer and W.Weinmann, Development and ValidaFon of an LC-MS/MS
method aCer chiral derivaFzaFon for the simultaneous stereoselecFve determinaFon of Risperiodone and 9-
Hydroxyrisperidone in human plasma, Anal. Chem, 2014, Volume 86, pages 1640-1647.
6 hVps://www.waters.com/webassets/cms/library/docs/720001680en.pdf - Accessed 16/11/2023
7 Jedrzej Wysocki, Michael W. Dong, LCGC North America, Volume 37, Issue 10, Pages 750-759
8 Shankarling et al. “Chromophores and Chromophoric acFvity: An Organic Chemistry PerspecFve”, Journal of Chemical
Educa6on, Volume 89, 2012, pages 1246-1249
9 J. Krishnegowda, R. Kp, N. Gowda, Indian Journal of Pharmaceu6cal Sciences, 76, 379-466
10 S E. Ukwueze, European Journal Pharmaceu6cal and Medical Research, Volume 8, 2021, Pages 27-31
11
C.Russo, L. Wyld, M.Da Costa Aubreu, C.S.Bury, C.Heaton, L.M.Cole and S.Francese, Non-invasive screening of
Breast cancer from fingerKp smears – a proof of concept study, Scien&fic Reports, Volume 13, 2023, page 3
12
Venter, A., Sojika, P.E. and Cooks, R.G., Droplet Dynamics and IonizaKon Mechanisms in DesorpKon
Electrospray IonisaKon Mass Spectrometry, Analy&cal Chemistry, 2006, Volume 78, Pages 8549-8555
13
E.c. Cabral and M.F Mirabelli, DesoprKon electrospray ionizaKon mass spectrometry: a rapid screening tool
for veterinary drug preparaKon and forensic samples from hormone crime invesKgaKons, Analy&cal methods,
2017, Volume 9, Pages 1174-1182
14
D.R. Ifa, N.E. Manicke, A.L. Dill and R.G Cooks, Forensic analysis of inks by imaging desorpKon electrospray
inionzaKon (DESI) mass spectrometry, analy&cal methods, 2010, Volume 2, pages 461-467.
15 Heather J. Walker, Advanes in Botanical Research, Volume 98, 2021, Pages 271-296
16 G. Astarita, S. Dhungana, B.Shrestha, E.C.Laiakis, Methods in Enzymology, Volume 636, 2020, Pages 93-108
17 J Larry Campbell, JC Yves Le Blanc, Bioanalysis, 2015, Volume 7, pages 853-856
18
Dr David Smith’s PowerPoint presentaKons on Ion mobility
19
X.L. Guan, I. Riezman, M.R.Wenk, H.Riezman, Methods in Enzymology, Volume 470, 2010, Pages 369-391
20
R.KosKaninen and G.J.deVos, MulKdimensional Liquid Chromatography in target-based metabolomics:
Method development and applicaKon in the analysis of isotopically labelled plant extracts, Anal. Chem.,
Volume 85, pages 2493-2499
21
hfps://www.tofwerk.com/advantages-Kme-of-flight-mass-spectrometry-over-quadrupole-ms/ (Accessed
14/11/2023)
22
hfps://www.emissionsanalyKcs.com/Kme-of-flight-mass-spectrometry (Accessed 14/11/2023)
23
M.W. Nielen, AnalyKcal Error ReducKon Using Single Point CalibraKon for Accuracy and Precise RouKne
PesKcide Measurement by TOF-MS, J.Am. Soc. Mass Spectrom, 2007, Volume 18, 1982-1993
24 Xianhua Zhang et al, Chromatographia, Volume 71, 2010, pages 1015-1023
25
E. de Hoffmann and V. Stroobant, Mass Spectrometry: Principles and ApplicaKons, 3rd
edn, John Wiley and
Sons, 2007
26
S. Eliuk and A. Makarov, EvoluKon of Orbitrap Mass Spectrometry InstrumentaKon, Annu. Rev. Anal. Chem.,
2015, Volume 8, pages 61-80.

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Analytical Coursework - Molly Winterbottom.pdf

  • 1. Devise a rapid and non-destructive mass spectrometric method to detect and quantify the presence of Risperidone and its major metabolite 9-Hydroxyrisperidone in a fingertip smear. By Molly Winterbo.om Table of Contents Abstract.............................................................................................................................1 Characteris*cs of Risperidone.....................................................................................................2 Previous techniques used ...........................................................................................................3 Prior to running the method .......................................................................................................4 Method..............................................................................................................................5 Instrumenta*on Requirements/sugges*ons: ..............................................................................5 DESI ...............................................................................................................................................................5 DMS...............................................................................................................................................................6 MRM..............................................................................................................................................................7 MS-TOF..........................................................................................................................................................7 Quan*fica*on of Results.............................................................................................................8 Alterna*ve future adapta*ons to the method.............................................................................9 Conclusion .........................................................................................................................9 References .........................................................................................................................9 Abstract The aim of this paper is to iden0fy and possibly develop an analy0cal chemistry approach to detect and quan0fy the presence of an an0psycho0c drug known as Risperidone and its major metabolite 9-Hydroxyrisperidone, present in a finger0p smear during a rou0ne Drug monitoring check. Usually, these checks would be carried out on the pa0ents’ blood plasma or urine sample. However, this paper gives way to a new route of carrying out this check, using a finger0p smear sample given by the pa0ents, which will provide an alterna0ve, faster and less invasive sample collec0on, which is also less likely to be tampered with by the pa0ents. This method combines the use of DESI (Desorp0on electrospray ionisa0on), DMS (Differen0al Mobility Spectrometry), MRM (Mul0ple-reac0on monitoring) where the sample will enter a triple quadrupole, this is then coupled with a MS-TOF (Time-of-Flight) tandem MS. Using this method will enable a non-destruc0ve analysis of the finger0p smear, with a possibility of detec0ng the enan0omers of Risperidone and it’s metabolite 9- Hydroxyrisperidone for further future analysis if required. Here, the details of each component and its benefit will be explained along with any alterna0ve method that could be used and any previous methods that have already been used when detec0ng the drug in blood plasma or urine.
  • 2. Characteris*cs of Risperidone Figure 1. Molecular structure of Risperidone As Risperidone is metabolised in the body it results in the forma0on of 2 enan0omers due to its chiral centre, (+)-9-Hydroxyrisperidone and (-)-9-Hydroxyrisperidone1 . Chirality plays a significant role in the detec0on and quan0fica0on of drug substances, however in the case of Risperidone the enan0omers are similar in their effects when administered to pa0ents2 so the desire to separate the metabolites using specific methods is not needed but this technique could be used for this purpose. Figure 2. Molecular Structure of 9-Hydroxylrisperidone, the metabolite of Risperidone Based off previous studies we can determine Risperidone’s m/z in a range of 411 to 191, its metabolite 9-Hydroxyrisperidone in the range of 427 to 2073 , therefore these will be the values set at the quadrupole in the MRM phase of the instrument to find the m/z peak and the peak of its product ion in order to help the quan0fica0on process at the end of the analysis. Due to the presence of heterocycles containing nitrogen and a piperidine ring, this indicates that the basic proper0es present in Risperidone makes it suitable for protona0on using DESI, more specifically the two benzisoxazole rings that contain nitrogen atoms will be protonated in a posi0ve ionisa0on mode4 . From previous studies on Risperidone and its analysis using mass spectroscopy also indicated that it would ionise under posi0ve ion condi0ons, producing the [M+H]+ ions.5
  • 3. Figure 3. Fragment ions that will produce a high intensity peak on the m/z spectra, Risperidone fragment ion on the Right and 9-Hydroxyrisperidone fragment ion on the LeC. Other fragment ions can be seen in the spectra, see figure 7 for the spectra along with the fragment ions or Figure 3 for the fragment ions. These fragments contain the change between Risperidone and 9-Hydroxyrisperidone due to the presence of an OH group a^er it has been metabolised. From looking at Risperidone and its metabolites structure, it presents polar characteris0cs due to it containing nitrogen, promo0ng electronega0vity in the compound, especially being encompassed in the heterocyclic aroma0c rings. So again, the use of posi0ve ionisa0on will occur. Previous techniques used The monitoring of Risperidone adherence in the body has been recorded previously in many studies using various analy0cal techniques including LC-MS/MS or HPLC/ MS/MS as the technique of choice, due to its fast, sensi0ve, and selec0ve analysis. However, these benefits can also be its downfall when it comes to the analysis of drugs such as Risperidone and its metabolite 9-hydroxyrisperidone. It’s fast paced analysis increases the probability of the sample of interest elu0ng from the column with another interfering compound mixed within the sample taken from pa0ents urine or blood plasma6 , or in this case fingerprint smear. In this paper, the aim is to devise a technique to be used to determine the detec0on and quan0fica0on of Risperidone and 9-Hydroxyrespirdone in a fingerprint smear given from pa0ents during a drug adherence examina0on without the use of HPLC to avoid the possibility of the analyte elu0ng from the column undetected. Many of these techniques paired their HPLC with UV detectors, which is common prac0se due to its ease of use and reliability. It’s benefits include the fact that it is a non-destruc0ve technique and that it can provide informa0on on peak iden0ty. However, in order to detect using UV the analyte must have chromophoric ac0vity7 , which is the presence of a chromophore group in the molecule that has the ability to absorb light energy in the visible spectrum8 . Risperidone is a non-chromophoric an0psycho0c compound, so UV-detec0on would not be suitable to quan0fy the presence in the finger0p smear of the pa0ents.
  • 4. Prior to running the method Before running any actual finger0p smear, the first thing would be to calibrate the machine using standards of Risperidone and its metabolite. This should be done in order to produce a calibra0on curve to enable effec0ve quan0fica0on of the finger0p smear at the end of the test. Standards such as Risperidone tablets can be used in varying doses such as 1,2 and 3mg tablets9 , this can then be used to create standard solu0ons with varying concentra0ons by dissolving and dilu0ng in solvents such as methanol, enabling droplets to be placed onto a glass microscope slide and run on the machine un0l the instrument starts to pick up and detect the standards, producing its peaks. Alongside the analysis of the standards the solvent, mobile and sta0onary phases would be altered to increase the peak detec0on. Figure 4. Example of a Standard calibraFon curve of Risperidone10 A^er a standard calibra0on curve has been produced (see Figure 4 for an example of a calibra0on curve), and reproduced for method valida0on, a sample of a finger0p smear can be taken from a pa0ent, disregarding which finger but making sure to take at least 3 per pa0ent to be able to provide reproducibility and run mul0ple fingerprints for the detec0on of Risperidone per pa0ent. It may also be beneficial to overlap all 3 finger0p smears on 1 slide to make sure enough of Risperidone is present in order for it to be detectable and quan0fiable. Another step to consider is sample prepara0on prior to placing on the DESI sample stage. A suggested technique to collec0ng the finger0p smears would be to have the pa0ents clean their hands in 100% ethanol solu0on in order to prevent further contaminants from being placed onto the glass slide with the finger0p smear11 . This step will help reduce peaks of non-interest to the chemist and help the selec0vity for trying to detect Risperidone and 9- Hydroxyrisperidone.
  • 5. Method Scheme 1. Flow chart of the suggested method Shown above is the layout of the suggested method that could poten0ally be used to analyse the presence of Risperidone and 9-hydroxyrisperidone in pa0ents finger0p smear. This paper will now discuss each component of this method in further detail along with explana0ons as to why this specific system has been chosen. Instrumenta*on Requirements/sugges*ons: The solvent suggested to use would be a ra0o of 95:5 v/v methanol: water with 1-5% ace0c acid modifier. However, if the user would like to op0mise the solvent use themselves, it would be done by star0ng off with a 50:50 v/v methanol: water working their way up to the 95:5 v/v along with adjus0ng the ace0c acid modifier from 0.1% up to the suggested 5% to see which works best and gives a peak with the best shape and intensity.12 The spray voltage can be monitored by varying its voltage range from around 4 to 6kV, this will promote efficient ion genera0on. The spray solvent flow will be measured in microL/min increments balancing out stability13 . Within the DESI source, the probe height an angle would need altering to op0mum coverage of the fingerprint smear, this would be done by adjus0ng the distance from the surface from around 1mm to 4mm and an angle from around 40 to 60 degrees.14 DESI Using a more non-destruc0ve method to start the analysis process like DESI, rather than MALDI, would allow liile upfront sample prepara0on which would also allow less tampering with the finger0p smear. Figure 5. Diagram of DESI system.15
  • 6. By taking the finger0p smear directly onto a glass microscope slide and placing it onto the automated moving stage under a DESI probe (See Figure 5), the DESI would be able to apply the charged solvent droplets (see instrumenta0on Requirements/sugges0ons for more informa0on) from the probe onto the sample, making sure to cover the en0re fingerprint smear, including any standards doied on the smear or around it on the edge of the glass slide, if the standard calibra0on is being run alongside the fingerprint smear analysis. The finger0p smear sample could be washed with water in a squeeze boile prior to running on DESI, to remove any compounds that could interfere with the detec0on of Risperidone, but if the pa0ents had washed their hands with the 100% alcohol first then it wouldn’t make much difference. Using water will not affect Risperidone’s detec0on as it has poor aqueous solubility. A key step to take when using DESI for ionisa0on is making sure to have con0nuous and op0mal scan of the fingerprint smear. In order to do this, the ion intensi0es of the Peak of interest (Risperidone, 9-hydroxyrisperidone and the standard) must be monitored by the adjustment of the electrospray incidence angle, the distance between the sample surface, emiier, and MS inlet capillary, also the distance between the sample surface and the ion collec0on capillary16 . DMS A^er the sample has been ionised, the ions will be carried away by Nitrogen, the carrier gas in DMS where they will be separated based on differences in ion mobility in an electric field, adding another dimension of separa0on to the analy0cal technique as the results will include Risperidone and its metabolite, so the more separa0on will be needed to avoid any peak overlaps. A separa0on voltage will force the ions to move closer to one electrode than the other planar electrode. To stop the ion of interest going to the electrode a counter voltage (compensa0on voltage or direct current voltage) is applied to the system17 . These voltages need to be altered and tweaked to only enable the ions of interest through to the first quadrupole in MRM. Figure 6. Image of how DMS ion mobility works.18 Since the sample will include Risperidone and its metabolite, there will be different mobility profiles, therefore using DMS coupled to DESI would improve selec0vity of detec0on. Along
  • 7. with the removal of any background noise in the spectra and providing cleaner peaks for further analysis. It will also provide rapid separa0on of Risperidone’s isomers if further analysis is wanted on the specific isomer men0oned in characteris0cs of Risperidone sec0on of this paper. MRM The use of MRM on a triple quadrupole mass spectrometer would allow the operator to select the target m/z for both Risperidone and 9-Hydroxyrisperidone allowing those ions with that mass to charge ra0o through to then be fragmented at the second quadrupole and from there a specific fragment can be selected at the third quadrupole. Based off of previous studies, the first quadrupole will be set to the condi0ons where Risperidone’s m/z in a range of 411 to 191, its metabolite 9-Hydroxyrisperidone in the range of 427 to 207, allowing only these ions through to be fragmented at the second quadrupole producing the fragment ions [C23H27FN4O2]+ for Risperidone and [C23H27FN4O3]+ for 9- hydroxyrisperidone, to which the third quadrupole would be set to only let these fragmented ions through to the TOF. Using MRM would be profoundly beneficial as it would provide highly sensi0ve and selec0ve quan0fica0on of the analyte of interest (Risperidone and its metabolite) whilst using it alongside internal standards that would be recognised by the database19 . This highly sensi0ve and selec0ve system will enable the analyte to be picks on its m/z value, therefore monitoring specific precursor and product ion transi0ons, resul0ng in a significant decrease of background noise and interferences20 . Although using MRM can be a costly method of choice, it may be the only suitable op0on to use due to its ability to carry out target analysis on the compound of interest. This con is then outweighed by the benefit of the system and the use it is intended for. MS-TOF Aiached to a single quadrupole for the ions to simple pass through, the high mass accuracy and resolu0on of TOF (Time-Of Flight) will enable accurate mass measurement of the precursor and fragment ions for each compound detected by MRM, allowing the confirma0on of the molecular formulas for both Risperidone and its metabolite. As a TOF mass analyser works by separa0ng the ions based on their velocity the assump0on is that the ions with the smaller m/z will move more quickly through the chamber of the TOF and be detected first21 . In this case, based on the informa0on stated, another assump0on can be made sugges0ng that the ion for Risperidone will hit the detector first as it has the smaller m/z value for the precursor and fragmented ions compared to the ions for 9- hydroxyrisperidone. Finishing the method by adding a MS-TOF will further increase the instruments sensi0vity in being able to detect the analytes of interest, along with its resolu0on and mass accuracy.22 However, using an MS-TOF has been known to have some limita0ons around ionisa0on source interac0ons, mass accuracy dri^ and data processing. So, during the trial, checks may have to be carried out to make sure the TOF analysers don’t dri^, this can be done with the use of internal standards. Looking at the data processing aspect, that is subjec0ve to the
  • 8. analyte in ques0on and what the user wants to analyse. The immense full spectral data produced by the MS-TOF can be overwhelming to the computer so^ware23 and the user, so it may be beneficial to look at an alterna0ve to MS-TOF like a triple quadrupole, Orbitrap or Ion Trap Mass Spectrometers. Quan*fica*on of Results From looking an example spectrum taken from a paper, Risperidone and 9- hydroxyrisperidone can be detected by finding its m/z peak and the product ion peak. Looking below at Figure 7 the spectra show Risperidone and its metabolite being detected at the values set at the triple quadrupole during MRM. Figure 7. Example spectra of Risperidone and 9-Hydroxyrisperidone24 By using the calibra0on curve produced from the standards of Risperidone, the concentra0on of the sample can be measured and quan0fied by finding the area of the peak produced in the sample MS spectrum and using a line of best fit along the graph and looking at the x-axis for the concentra0on value. See Figure 4 for an example of a calibra0on curve produced from a Risperidone standard. Since Risperidone and its metabolite aren’t a protein, it would not work to perform a de novo analysis on the spectra, so the best way to quan0fy the results would be to compare using a calibra0on curve that was produced on the same machine using a standard of
  • 9. risperidone and possibly making another curve for a standard of its metabolite to use that for quan0fica0on on 9-hydroxyrisperidone. Alterna*ve future adapta*ons to the method Stated in the DESI sec0on of this paper an alterna0ve to using this ionisa0on technique would be to use MALDI (Matrix-Assisted Laser Desorp0on/ionisa0on), although this method would be more destruc0ve on the sample as it uses a UV laser to transfer energy to the matrix. Another alterna0ve in this instrumenta0on method would be the use of DIA (Data- independent Acquisi0on) coupled with Dri^ mobility in place of MRM and DMS, although Dri^ mobility would have to occur a^er DIA and the quadrupole in order to separate the parent and daughter ion peaks in the spectra. As discussed previously in the MS-TOF sec0on of this paper, an alterna0ve to use for the detec0on of the precursor and fragment ions would be a triple quadrupole, Ion trap or Orbitrap Mass Spectrometers. The use of a Triple Quadrupole would enable superior quan0ta0ve accuracy and precision when targe0ng specific analytes, however, with this proposed technique the incorpora0on of a triple quadrupole has already been installed with the use of MRM25 . Another alterna0ve could be the use of an Orbitrap. The Orbitrap could be considered very similar to the MS-TOF analyser as it also offers high mass accuracy and resolu0on26 . Conclusion In conclusion, the development of the method incorpora0ng the components of DESI-DMS- MRM-MS-TOF has been produced based off of previous studies done on the detec0on and quan0fica0on of Risperidone and its metabolite 9-hydroxyrisperidone in pa0ent’s blood plasma or urine or the comparison of different tablets of Risperidone produced by various manufacturers in different countries, the main focus on those papers being the standardisa0ons techniques used. Although in theory this method will work in producing detectable and quan0fiable ions of the analyte of interest, the only way to jus0fy and thoroughly validate this method would be trialling it in a lab under the right condi0ons, with suitable and willing pa0ents, carrying out mul0ple robust tes0ng with a wide range of finger0p smears. References 1 Michael G. Aman, Et al, Clin Ther. 2007 Jul, 29, 1476-1486 2 M.C. Mauri, S. PaleVa, M.Maffini, Excli Journal, 2014, 13, 1163-1191 3 D E. Moody, J D. Laycock, W. Huang, and R L. Foltz, Journal of Analy6cal Toxicology, Vol 28, September 2004 4 R.G. Cooks, Z. Ouyang, Z. Takats and J.M. Wiseman, Ambient Mass Spectrometry, Science, 2006, Volume 311, Pages 1566- 1570. 5 A.E. Steuer, A.B. Poetzch, M.R.Baumgartner, S.D Kraemer and W.Weinmann, Development and ValidaFon of an LC-MS/MS method aCer chiral derivaFzaFon for the simultaneous stereoselecFve determinaFon of Risperiodone and 9- Hydroxyrisperidone in human plasma, Anal. Chem, 2014, Volume 86, pages 1640-1647. 6 hVps://www.waters.com/webassets/cms/library/docs/720001680en.pdf - Accessed 16/11/2023 7 Jedrzej Wysocki, Michael W. Dong, LCGC North America, Volume 37, Issue 10, Pages 750-759 8 Shankarling et al. “Chromophores and Chromophoric acFvity: An Organic Chemistry PerspecFve”, Journal of Chemical Educa6on, Volume 89, 2012, pages 1246-1249 9 J. Krishnegowda, R. Kp, N. Gowda, Indian Journal of Pharmaceu6cal Sciences, 76, 379-466
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