This document describes a spectrofluorometric method for determining two cephalosporin drugs, cefadroxile and cefuroxime sodium, in pharmaceutical formulations. The method involves reacting the drugs with 1,2-naphthoquinone-4-sulfonate under alkaline conditions to form fluorescent derivatives, extracting them with chloroform, and measuring fluorescence intensity. The method was optimized and validated, demonstrating good linearity, accuracy, precision and sensitivity for quantifying the drugs within certain concentration ranges in samples. The effects of various parameters on the analysis were also examined.

1. SPECTROFLUOROMETER
Presented by: Nour Elrashed
Q.C master student
Damascus UniversityFaculty of Pharmacy

2. CONTENTS:
 Introduction
 the principle of spectrofluorometer
 What can specrofluorometer do?
 The components of specrofluorometer
 Fluorescence Spectra
 Fluorescence and chemical structure
 Applications of Spectrofluorometer
 New pharmaceutical studies conducted by
spectrofluorometer
 References

3. Emission
Molecular
Photoluminescence
Fluorescence
spectrofluorometer
Phosphorescence
Atomic
Absorption

4. HISTORY:
 The term fluorescence comes from the mineral
fluorspar (calcium fluoride) when Sir George G.
Stokes observed in 1852 that fluorspar would give
off visible light (fluoresce) when exposed to
electromagnetic radiation in the ultraviolet
wavelength.

5. THE PRINCIPLE OF SPECTROFLUOROMETER:
 It is an analytical device depends on the fluorescence
phenomenon which is a short-lived type of
photoluminescence created by electromagnetic excitation.
 That is, fluorescence is generated when a molecule
transmits from its ground state So to one of several
vibrational energy levels in the first excited electronic
state, S1, or the second electronic excited state, S2, both
of which are singlet states.
 Relaxation to the ground state from these excited states
occurs by emission of energy through heat and/or
photons.

6.  The difference between the excitation and emission
wavelengths is called the Stokes shift.
 Stokes’ studies of fluorescent substances led to the
formulation of Stokes’ Law, which states that the
wavelength of fluorescent light is always greater
than that of the exciting radiation. Thus, for any
fluorescent molecule, the wavelength of emission is
always longer than the wavelength of absorption.

8. WHAT IS THE FLUORESCENCE QUANTUM YIELD (ΦF)?
 It is the quantitative expression of the fluorescence
efficiency, which is the fraction of excited molecules
returning to the ground state by fluorescence.
 Quantum yields range from 1, when every molecule in
an excited state undergoes fluorescence, to 0 when
fluorescence does not occur.

9.  A molecule’s fluorescence quantum yield is
influenced by external Variables such as:
• temperature
• viscosity of solvent
• pH
 Increasing temperature generally decreases Φf
because more frequent collisions between the molecule
and the solvent increases external conversion.
 Decreasing the solvent’s viscosity decreases Φf for
similar reasons.
 For an analyte with acidic or basic functional groups, a
change in pH may change the analyte’s structure
and, therefore, its fluorescent properties.

10.  Emission of a photon
when the analyte
returns from a higher-
energy state to a lower-
energy state with the
opposite spin (triplet
excited state: An
excited state in which
unpaired electron spins
occur.).
 Average lifetime from
10-3 to 10+2 sec
 Emission of a photon
when the analyte
returns from a higher-
energy state to a lower-
energy state with the
same spin (singlet
excited state: An
excited state in which
all electron spins are
paired).
 Average lifetime from
10-9 to 10-7 sec
Phosphorescence Fluorescence

13. WHAT CAN SPECROFLUOROMETER DO?
 It has been used for the direct or indirect quantitative and
qualitative analysis by measuring the fluorescent
intensity F.
 It is relatively inexpensive and sensitive (the sensitivity
of fluorescence is approximately 1,000 times greater
than absorption spectrophotometric methods).

14.  fluorescent intensity F is dependent on both intrinsic
properties of the compound (fluorescence quantum yield
Φf), and on readily controlled experimental parameters
including:
• intensity of the absorbed light I0
• molar absorption coefficient Ɛ
• path length of the cell b
• concentration of the fluorophor in solution c

15.  At low concentrations of fluorophore, the fluorescence
intensity of a sample is essentially linearly proportional
to concentration.
 However, as the concentration increases, a point is
reached at which the intensity increase is progressively
less linear, and the intensity eventually decreases as
concentration increases further.

16. fluorescence intensity
concentrations

17.  The most common reason for this is Inner filter effect
that, as the absorbance of the sample increases, the
light intensity experienced by some of the fluorescent
molecules is lower than that experienced by others.
When excitation intensity decreases, so does
fluorescence emission intensity.
 It is generally necessary to use concentrations that
result in absorbance values of 0.1 or lower to observe
concentration dependent emission.

18.  As the concentration of molecules in a solution
increases, probability increases that excited molecules
will interact with each other and lose energy through
processes other than fluorescent emission. Any process
that reduces the probability of fluorescent emission is
known as quenching.
 Other parameters that can cause quenching include:
• presence of impurities
• increased temperature
• reduced viscosity of the solution media

19. THE COMPONENTS OF SPECROFLUOROMETER:
1. light source (75 to 450-W high-pressure xenon arc
lamp or Lasers)
2. excitation monochromator
3. sample holder (Quartz/Optical Glass/Plastic Cells)
4. emission monochromator
5. Detector (photomultiplier)
6. Most spectrofluorometers also have a reference
sample. The reference is generally a solution of a
strongly fluorescent molecule with a broad
absorbance spectrum such as rhodamine. The
reference is necessary to correct for lamp
output, especially when varying the excitation
wavelength, and to correct for differences in
detector sensitivity.

22. FLUORESCENCE SPECTRA:
 Photoluminescence spectra are recorded by measuring
the intensity of emitted radiation as a function of either
the excitation wavelength or the emission wavelength.
 The excitation spectra is determined by measuring the
emission intensity at a fixed wavelength , while varying
the excitation wavelength. It is useful for selecting the
best excitation wavelength for a quantitative or
qualitative analysis.
 The emission spectra is determined by measuring the
variation in emission intensity wavelength for a fixed
excitation wavelength.

24. FLUORESCENCE AND CHEMICAL STRUCTURE:
 Fluorescence is generally observed with molecules
where the lowest energy absorption is →*
transition, although some n→* transitions show weak
fluorescence.
 Most unsubstituted, nonheterocyclic aromatic
compounds show favorable fluorescence quantum
yields wich usually increases with the number of rings
and their degree of condensation. In
addition, substitution to the aromatic ring can have a
significant effect on Φf. For example, the presence of an
electron-withdrawing group, such as (NO2), decreases
Φf, whereas adding an electron-donating group, such as
(OH), increases Φf.

25.  Fluorescence also increases for aromatic ring
systems and for aromatic molecules with rigid planar
structures.
 The simple heterocyclics, such as
pyridine, furan, thiophene, and pyrrole do not exhibit
fluorescence; on the other hand, fused ring structures
ordinarily do.

26. SPECTROFLUOROMETER:APPLICATIONS OF
 Environmental Significance:
 To detect environmental pollutants such as polycyclic
aromatic hydrocarbons:
• pyrene
• benzopyrene
• organothiophosphorous pesticides
• carbamate insecticides
 Geology:
 Many types of calcite and amber will fluoresce under
shortwave UV. Rubies, emeralds, and the Hope
Diamond exhibit red fluorescence under short-wave UV
light; diamonds also emit light under X ray radiation.

27. SPECTROFLUOROMETER:APPLICATIONS OF
 Analytical chemistry
 to detect compounds from HPLC flow
 TLC plates can be visualized if the compounds or a
coloring reagent is fluorescent
 Biochemistry:
 used generally as a non-destructive way of tracking or
analysis of biological molecules (proteins)
 Possible direct or indirect analysis aromatic amino acids
(phenylalanine- tyrosine-tryptophan)
 Fingerprints can be visualized with fluorescent
compounds such as ninhydrin.

28. SPECTROFLUOROMETER:APPLICATIONS OF
 Medicine
 Blood and other substances are sometimes detected by
fluorescent reagents, particularly where their location was not
previously known.
 There has also been a report of its use in differentiating
malignant, bashful skin tumors from benign.
 Pharmacy:
 Possible direct or indirect analysis drugs such as:
• vitamins (vitamin A -vitamin B2 -vitamin B6 -vitamin B12 -
vitamin E -folic acid)
• catecholamines (dopamine-norepinephrine)
• Other drugs (quinine-salicylic acid–morphine-barbiturates –
LSD)

29. NEW PHARMACEUTICAL STUDIES CONDUCTED
BY SPECTROFLUOROMETER:

30. OPTIMIZATION AND VALIDATION OF SPECTROFLUORIMETRIC METHOD FOR
DETERMINATION OF CEFADROXILE AND CEFUROXIME SODIUM IN
PHARMACEUTICAL FORMULATIONS
.HYNEINE-BOULA,SMHMEDA,AALBASHIRE
CHEMISTRY DEPARTMENT, FACULTY OF SCIENCE, UNIVERSITY OF KHARTOUM, PO BOX 321, KHARTOUM, 11115, SUDAN.
 Abstract
A simple, accurate, precise and validated spectrofluorimetric method is proposed for
the determination of two cephalosporins, namely, cefadroxile (cefa) and cefuroxime
sodium (cefu) in pharmaceutical formulations. The method is based on a reaction
between cephalosporins with 1,2-naphthoquinone-4-sulfonate in alkaline medium, to
form fluorescent derivatives that are extracted with chloroform and subsequently
measured at 610 and 605 nm after excitation at 470 and 460 nm for cefa and cefu
respectively. The optimum experimental conditions have been studied. Beer's law is
obeyed over the concentrations of 20-70 ng/mL and 15-40 ng/mL for cefa and
cefu, respectively. The detection limits were 4.46 ng/mL and 3.02 ng/mL with a linear
regression correlation coefficient of 0.9984 and 0.998, and recoveries ranging 97.50-
109.96% and 95.73-98.89% for cefa and cefu, respectively. The effects of
pH, temperature, reaction time, 1,2-naphthoquinone-4-sulfonic concentration and
extraction solvent on the determination of cefa and cefu, have been examined. The
proposed method can be applied for the determination of cefa and cefu in
pharmaceutical formulations in quality control laboratories. Copyright © 2013 John
Wiley & Sons, Ltd.
ahead of print]Epub. [2481/bio.1002.10:doi.24Jan2013Luminescence.

31. cefadroxile
1,2-naphthoquinone-4-sulfonate
cefuroxime sodium

32. A HIGHLY SENSITIVE FLUORIMETRIC METHOD FOR DETERMINATION
VIACAPSULESANDFORMBULKITSINLENALIDOMIDEOF
DERIVATIZATION WITH FLUORESCAMINE
.NZLZOMANA,AHAKHEITB,NYHALILK,IAARWISHD
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY, COLLEGE OF PHARMACY, KING SAUD UNIVERSITY,, P,O, BOX 2457, RIYADH, 11451, SAUDI
ARABIA. IDARWISH@KSU.EDU.SA.
ABSTRACT:
BACKGROUND: Lenalidomide (LND) is a potent novel thalidomide analog which demonstrated remarkable
clinical activity in treatment of multiple myeloma disease via a multiple-pathways mechanism. The strong
evidences-based clinical success of LND in patients has led to its recent approval by US-FDA under the trade
name of Revlimid® capsules by Celgene Corporation. Fluorimetry is a convenient technique for
pharmaceutical quality control, however there was a fluorimetric method for determination of LND in its bulk
and capsules.
RESULTS: A novel highly sensitive and simple fluorimetric method has been developed and validated for the
determination of lenalidmide (LND) in its bulk and dosage forms (capsules). The method was based on
nucleophilic substitution reaction of LND with fluorescamine (FLC) in aqueous medium to form a highly
fluorescent derivative that was measured at 494 nm after excitation at 381 nm. The factors affecting the
reaction were carefully studied and optimized. The kinetics of the reaction was investigated, and the reaction
mechanism was postulated. Under the optimized conditions, linear relationship with good correlation
coefficient (0.9999) was found between the fluorescence intensity and LND concentration in the range of 25-
300 ng/mL. The limits of detection and quantitation for the method were 2.9 and 8.7 ng/mL, respectively. The
precision of the method was satisfactory; the values of relative standard deviations did not exceed 1.4%. The
proposed method was successfully applied to the determination of LND in its bulk form and pharmaceutical
capsules with good accuracy; the recovery values were 97.8-101.4 ± 1.08-2.75%.
CONCLUSIONS: The proposed method is selective and involved simple procedures. In conclusion, the method is
practical and valuable for routine application in quality control laboratories for determination of LND.
.118-6-X153-1752/1186.10:doi.118):1(6;16Oct2012Cent J.Chem

33. lenalidomide
fluorescamine

34. INTIZANIDINEOFDETERMINATIONSPECTROFLUORIMETRICENSITIVES
PHARMACEUTICAL PREPARATIONS, HUMAN PLASMA AND URINE
THROUGH DERIVATIZATION WITH DANSYL CHLORIDE
.STLUU
DEPARTMENT OF ANALYTICAL CHEMISTRY, FACULTY OF PHARMACY, ISTANBUL UNIVERSITY, TURKEY. SEVGITATAR@YAHOO.COM
 Abstract
A sensitive spectrofluorimetric method was developed for the determination of
tizanidine in human plasma, urine and pharmaceutical preparations. The method
is based on reaction of tizanidine with 1-dimethylaminonaphthalene-5-sulphonyl
chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent
derivative that was measured at 511 nm after excitation at 383 nm. The different
experimental parameters affecting the fluorescence intensity of tizanidine was
carefully studied and optimized. The fluorescence-concentration plots were
rectilinear over the ranges 50-500 and 20-300 ng/mL for plasma and
urine, respectively, detection limits of 1.81 and 0.54 ng/mL and quantification
limits of 5.43 and 1.62 ng/mL for plasma and urine, respectively. The method
presents good performance in terms of linearity, detection and quantification
limits, precision, accuracy and specificity. The proposed method was successfully
applied for the determination of tizanidine in pharmaceutical preparations. The
results obtained were compared with a reference method, using t- and F-tests.
.28Oct2011Epub.1367/bio.1002.10:doi.30-426):5(27Oct;-Sep2012Luminescence.

35. dansyl chloride
tizanidine

36. INHCLPAROXETINEOFDETERMINATIONPECTROFLUORIMETRICS
PHARMACEUTICALS VIA DERIVATIZATION WITH 4-
CHLORO-7- NITROBENZO-2-OXA-1,3-DIAZOLE (NBD-CL)
.HLMANSIE,NNANYE-LE,FELALB,MALSHW
DEPARTMENT OF ANALYTICAL CHEMISTRY, FACULTY OF PHARMACY, UNIVERSITY OF
MANSOURA, MANSOURA, EGYPT.
 Abstract:
A sensitive and simple spectrofluorimetric method has been developed and
validated for the determination of the antidepressant paroxetine HCl (PXT) in its
dosage forms. The method was based on coupling reaction of PXT with 4-
chloro-7-nitrobenzo-2- oxa-1,3-diazole (NBD-Cl) in an alkaline medium (pH 8)
to form a highly fluorescent derivative that was measured at 530 nm after
excitation at 460 nm. The factors affecting the formation and stability of the
reaction product were carefully studied and optimized. The fluorescence-
concentration plot is rectilinear over the range 0.2-6 μg/mL with LOD of 0.08
μg/mL and LOQ of 0.24 μg/mL respectively. The method was applied to the
analysis of commercial tablets and the results were in good agreement with
those obtained using the reference method. The mean percentage recoveries
for paxetin and xandol tablets were 101.27 ± 1.79 and 101.33 ± 1.19
respectively. A proposal of the reaction pathway was postulated.
J Fluoresc. 2011 Jan;21(1):105-12. doi: 10.1007/s10895-010-0693-2. Epub 2010 Jul 1.

37. paroxetine
4-chloro-7-nitrobenzo-2- oxa-1,3-diazole

38. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC AND
SPECTROFLUOROMETRIC DETERMINATION OF ΑLPHA- TOCOPHEROL IN A
NATURAL PLANT: FERULA HERMONIS (ZALOOH ROOT)
CANNAHHALILK,BHIDIACCHIWAG,,BIMARAMILJ,AOUNAMILEE
A LEBANESE AGRICULTURAL RESEARCH INSTITUTE (LARI), FANAR, P.O. BOX 90 1965, JDEIDET EL-METN, LEBANON
B LABORATORY OF MOLECULAR CHEMISTRY, FACULTY OF SCIENCE II, THE LEBANESE UNIVERSITY, FANAR, P.O. BOX
26110217 FANAR-MATN, LEBANON
 Abstract:
A high-performance liquid chromatographic (HPLC) method for the determination of α-
tocopherol in a natural plant (Ferula hermonis–Zalooh roots) is reported. The method
includes saponification of samples and extraction of α-tocopherol with a mixture of
acetonitrile and methanol (1:1 v/v). However, the presence of α-tocopherol in Zalooh
is confirmed with HPLC-UV and fluorescence detection. A spectrofluorometric and the
internal addition standard methods are also used to quantify the α-tocopherol in the
plant. An internal standard method is based on a known concentration of α-tocopherol
that is added in every sample that is analyzed. Alpha-tocopherol levels as determined
in samples by HPLC with UV and fluorescence detection did not differ significantly
from the levels determined by Shimadzu spectrofluorometer . However, the amount of
tocopherol determined by both techniques in Zalooh roots was relatively very high.
Standards were checked for linearity giving correlation coefficients that were higher
than 0.99 in the concentration range of 1 and 6 μmol L−1.
615–607, Pages2005November,7, Issue18Volume

39. α-tocopherol

40. REFERENCES:
 www.pubmed.com
 www.sciencedirect.com
 Harvey.D, 2000, Modern Analytic Chemistry.
 Fluorescence and Fluorescence Applications-
Integrated DNA Technologies-2010
 Brandt.M, 2010, Spectrofluorometers and
Fluorescence Phenomena