CHM 260

BASIC INSTRUMENTALANALYSIS

TITLE OF ASSIGNMENT : HYPHENATED

INSTRUMENTATION TECHNIQUE AND
THEIR APPLICATION

NAME : NURSYAHIRAH BINTI SUHAILI

STUDENT ID :2015214946
GROUP : AS1205A2
LECTURER NAME : MADAM JULENAH AG
NUDDIN
DATE OF SUBMISSION : 12/10/2017
TITLE: HYPHENATED TECHNIQUES AND THEIR APPLICATION

INTRODUCTION

Hyphenated techniques combine chromatographic and spectral methods to exploit the

advantages of both. Chromatography produces pure or nearly pure fractions of
chemical components in a mixture. Spectroscopy produces selective information for
identification using standards or library spectra.(Kalpesh N Patel,2010) .
The term hyphenation was first adapted by Hirschfeld in 1980 to describe a possible
combination of two or more instrumental analytical methods in a single run
(Hirschfeld, 1980).
The power of combining separation technologies with spectroscopic techniques has
been demonstrated over the years for both quantitative and qualitative analysis of
unknown compounds in complex natural product extracts or fractions. There are many
types of hyphenated techniques, include GC-MS, LC-MS, LC-NMR, EC-MS, CE-MS,
GC-IR, LC- MS-MS, GC-MS-MS, GC-GC-MS, GC-NMR, and GC-AES.
TECHNIQUE : LC-MS ( LIQUID CHROMATOGRAPHY - MASS
SPECTROMETRY )

LC-MS is a chemistry technique that combines the physical separation of liquid

chromatography (or HPLC) with the mass spectroscopy. A typical automated LC-MS
system (Figure 1) consists of double three-way diverter inline with an autosampler,
LC system, the Mass spectrometer. The diverter generally operates as an automatic
switching valve to divert undesired portions of eluting from the LC system to waste
before the sample enters the MS.(Pavia et al., 2001)

FIGURE 1 : SCHEMATIC DIAGRAM FOR LC-MS

An LC-MS combines the chemical separating power of LC with the ability of an MS

to selectively detect and conrm molecular identity. MS is one of the most sensitive
and highly selective methods of molecular analysis, and provides information on the
molecular weight as well as the fragmentation pattern of the analyte molecule. The
information obtained from MS is invaluable for conrming the identities of the
analyte molecules.This qualitative analysis makes it possible to reconstruct an
unknown compound from MS data.
In LC operation for LC-MS, the preferred option is a reversed-phase system using a
gradient or isocratic solvent mixture of water, ACN, or MeOH. Small amounts of
acetic acid, formic acid, ammonium hydroxide/ammonia solution, or ammonium
acetate can also be used in the mobile phase.(Kalpesh N Patel,2010)
LC-MS is mainly separated into the three parts chromatography, interface and
spectrometry. In liquid chromatography separation is performed which is detected
with the help of Photo diode array, Ultraviolet, fluorescent like detectors. These
separated components then transferred to the interface. In interface the liquid is
volatilized and transferred to the MS. The LC-MS instrument can be interfaced by
electrospray, particle beam and thermospray. In addition , Electrospray is the most
widely used among the others interfaced . The spray needle is used as bridge to
connect the liquid chromatography with that of the mass. But the separate emitter is
flexible as well as convenient Anal (JP et al. Chem 2001) .
With the help of various ionization techniques the compound is ionized and then it is
analyzed by mass analyzer. Various mass analyzers are used include viz Quadrupoles,
quadrupole ion traps, time-to-flight (TOF), timeto-flight reflection (TOFR), and ion
cyclotron resonance (ICR) mass analyzers.

APPLICATION OF LIQUID CHROMATOGRAPHY-MASS

CHROMATOGRAPHY (LC-MS) .

One of the application of LC-MS is Therapeutic Drug Monitoring and Toxicology.

Dissatisfaction with the high cost of commercial immunoassays used in therapeutic
drug monitoring and their variable cross-reactivity with metabolites has spurred the
development of LC-MS assays as alternatives. Assays have been developed for
immunosuppressants such as cyclosporin, tacrolimus, sirolimus, everolimus and
mycophenolic acid, and this area has been reviewed .(Taylor PJ ,2004)
LC-MS assays for aminoglycosides , antiretroviralsand anticancer drugs have also
been developed.

The capacity to multiplex LC-MS assays so that several drugs and metabolites can be
measured in one run is a useful feature of these assays, which can simplify laboratory
workflows and provide additional pharmacokinetic information.
The use of LC-tandem MS for toxicology screening is attractive because of its
potential, relative to GC-MS screening, to provide greater confidence in
identifications, detect a wider range of drugs, toxins and their metabolites, and to
simplify sample preparation.
However, there are some issues that currently limit its widespread adoption as a
routine technique. Product ion spectra can vary considerably between instruments and
comprehensive spectral libraries are not available. Another limitation is that, unlike
GC-MS, there is no common set of MS operating conditions that is suitable for most
analytes. Different analytes have different optimal parameters (precursor ion, ion
source voltages, collision energies, etc.) which usually need to be determined on a
case-by-case basis. Most LC-MS toxicology strategies therefore rely on developing a
targeted panel containing as many as 301 drugs and metabolites.(Mueller CA,
Weinmann W, Dresen S, Schreiber A, Gergov M ,2005)
Data-dependent acquisition and rapid product ion scans using a set of different
conditions can assist this process but add to the complexity of the analysis.

Several LC-MS strategies for clinical and postmortem toxicology screening have been
published,(Pavlic M, Libiseller K, Oberacher H ,2006) and the field has been
reviewed.(Marquet P , 2002)
Direct analysis of urine samples is possible. Some potential pitfalls and
mis-identifications have been noted but these can be eliminated by carefully matching
retention times and mass spectra to a standard and using qualifier ion ratios. The
sensitivity of current instruments also allows the analysis of oral fluids and hair
samples

CONCLUSION
The advancement of hyphenated techniques, high resolution mass analyzers as well as
high throughput separation approaches, quantitative and quantitative analysis of
pharmaceutical drugs and metabolites can be achieved with good sensitivity.
REFFERENCE

1. Kalpesh N.Patel , Patel Jayvadan , Manish P. Patel & Hitesh A Patel .

(2010) .Introduction to hyphenated techniques and their applications in
pharmacy.Pharmaceutical Method ,1-11.DOI: 10.4103/2229-4708.72222
2. K.L. Lynch .(2017) . Toxicology: liquid chromatography mass
spectrometry .Retrieved from
http://www.sciencedirect.com/topics/neuroscience/liquid-chromatographymass-s
pectrometry
3. Susha Cheriyedath . (2016) . Liquid Chromatography-Mass Spectrometry
(LC-MS) Applications .Retrieved from
https://www.news-medical.net/life-sciences/Liquid-Chromatography-Mass-Spect
rometry-(LC-MS)-Applications.aspx
4. Hirschfeld, T. (1980). The Hyphenated Methods. Analytical Chemistry, Vol. 52,
No. 2 (February 1980), pp. 297A-312A, ISSN 0003-2700
5. Skoog DA, Holler FJ, Crouch SR (2007). Principles of Instrumental Analysis. 6th
Edition. Brooks/Cole Cengage Learning, Chapters 11, 20, 26, 27.
6. Pavlic M, Libiseller K, Oberacher H. Combined use of ESI-QqTOF-MS and
ESI-QqTOF-MS/MS with mass-spectral library search for qualitative analysis of
drugs. Anal Bioanal Chem. 2006;386:6982.
7. Taylor PJ. Therapeutic drug monitoring of immunosuppressant drugs by
high-performance liquid chromatography-mass spectrometry. Ther Drug
Monit. 2004;26:2159.
8. Maurer HH. Current role of liquid chromatography-mass spectrometry in clinical
and forensic toxicology. Anal Bioanal Chem. 2007;388:131525.
9. Mueller CA, Weinmann W, Dresen S, Schreiber A, Gergov M. Development of a
multi-target screening analysis for 301 drugs using a QTrap liquid
chromatography/tandem mass spectrometry system and automated library
searching. Rapid Commun Mass Spectrom. 2005;19:13328