University of Santo Tomas

Senior High School – Health Allied Strand

PRACTICAL RESEARCH 2 CHECKLIST FOR THE 1st QUARTER
Title (12 words Vanillin-Perchloric Acid Paper Chromatogram in Monitoring Testing
APA): Ovulation of Women Ages 18-25
Section: 12HA-3 Date: October 7, 2017 Overall Score:

Cuevas, Ma. Alexa Regina E.

Leader:
Batac, Franchesca C.
Nunez, Keziah Mae T. D.
Reyes, Mikaella Louisse D.
Members:
Lui, John Patrick C.

Put √ or
Documents Submitted Page no.
N/A
✓ 2-12
Practical Research 1 Capsule
✓ 13
Permit to conduct study/ Letter asking permission
✓ 14
Consent to participate in the study
✓ 15-20
Research Methods
N/A
Survey Questionnaire
N/A
Permit/Letter to use survey
✓ 21-24
Bibliography
✓ 25
Timetable
✓ 26
Budgetary Requirements
✓ 27-31
Curriculum Vitae

Page 1 of 31
 Pontifical and Royal
UNIVERSITY OF SANTO TOMAS
THE CATHOLIC UNIVERSITY OF THE PHILIPPINES

Senior High School

Capsule Research Proposal Form – STEM / HA Strand

Proponents: Batac, Franchesca Cunan

Cuevas, Ma. Regina Alexa Ebron

Lui, John Patrick Cajusay

Nuñez, Keziah Mae Tiu Dy

Reyes, Mikaella Louisse Dimalanta

Project Title: Use of Vanillin Perchloric Acid Paper Chromatogram testing in

monitoring ovulation of women ages 18-40 during their monthly
menstrual cycles as means to aid or avoid pregnancy

Project Discipline Cluster The University of Santo Tomas have five research flagships namely:

Page 2 of 31
 (Shade all that apply): ● Science and Technology
● Health & Allied Sciences
□ Social Sciences and Education
□ Arts and Humanities
□ Sacred Sciences

Among the areas of research in the

university are: □ Climate Change Preparedness
□ Natural products □ Education
● Chemical Sensors □ Arts
□ Biosensors □ Culture
□ Materials Science □ Humanities
□ Energy research □ Gerontology
□ Pure and Applied Microbiology □ Migration Studies
□ Biodiversity □ Youth Studies
□ Environmental Science □ Corporate Social Responsibility
□ Food Science and Engineering □Other,specify:
□ Urban Environment _________________________

Proposal Abstract: (Briefly summarize objectives, issues to be addressed, methods, and expected output.
Min-Max no. of words = 150-200)

For women trying to achieve or avoid pregnancy, being able to determine the
precise time of ovulation is important. There are different methods of detecting ovulation
such as, charting basal temperature, measuring LH and estrogen levels, and tracking
menstrual cycle. However, these methods are costly and are based on irregular factors
like body temperature and irregular menstrual cycle. Thus, this research aims to
investigate the viability of developing a low-cost test kit using Vanillin Perchloric acid
paper chromatogram test which monitors progesterone changes in a woman's ovulation
cycle. As progesterone is a more accurate indicator of a woman’s fertility, this device
would yield a more accurate result.

This study makes use of a mixed method paradigm which involves interviewing
gynecologists and testing the device. A select group of women ages 18-40 will test the
device for one menstrual cycle at the same time they will be tested at a clinic. Results
from the device will then be compared to the results from the clinical tests to ensure
accuracy and viability of the test kit.

Objectives: (Indicate general and specific objectives that needs to be addressed by the project. Min-
Max no. of words = 30-100)

The objective of this research is to prove the viability of using Vanillin

Perchloric acid paper chromatography test in measuring progesterone levels to monitor
ovulation of women ages 18-40. It will create a litmus type device that can be used as
“home kits” for couples to monitor ovulation as a means to achieve or avoid pregnancy. It
aims to check its effectivity, accuracy and price viability as compared to charting basal
body temperatures and measuring estrogen levels in clinics. It also aims to prove the
reliability of using progesterone in determining ovulation, as it is the least used method.

Conceptual Framework:

Page 3 of 31
Significance: (State the issues the research aims to address, and the significance of this research
project in relation to other researches on the same topics/ consider social and
technological impact of the research project. Min-Max no. of words = 50-100)

The findings of this study would benefit women to have better control of
achieving or avoiding pregnancy sans the inconvenience of laboratory testing. With
Vanillin Perchloric Acid Paper Chromatography Testing, women can have a testing
device that is highly accurate and comparatively cheap. The community would also
benefit using this device by providing a cheaper alternative to clinical tests of determining
ovulation.

Furthermore, future studies using the same chromatogram kits can be done for
hormonal monitoring of possible gallbladder problem and thyroid dysfunctions.

Page 4 of 31
Literature (Related works or studies in the research area. Min-Max no. of words = 800-1000)
Review:
Nature of ovulation

Ovulation process starts in the ovaries of a female wherein dominant follicle

secretes high levels of estrogen causing the release of the luteinizing hormone from the
pituitary gland. This hormone then triggers ovulation. Afterwards, the dominant follicle
becomes a corpus luteum and secretes progesterone. Since progesterone is released
only after ovulation, in retrospect, it can confirm the occurrence of the said event. The
same goes with urinary metabolite and pregnanediol 3-glucuronide, the substances
produced after the breakdown of progesterone.

Echochard and Gougeon (2000) aimed to figure out whether the female ovulation
affects the hormonal ovulation cycle within their bodies. The women observed were
normal having two intact ovaries with menstrual cycles averaging from 24-34 days. Upon
measurement of the cycle through trans-vaginal and trans-abdominal ultrasounds, they
found out that the progesterone secreted were not related to the ovulation contractions
observed, and that the follicle growth had not shown any difference between the three
phases observed in the study. Therefore, the hormonal changes within a woman cannot
be directly linked to her ovulation cycle.

In Scutt and Manning’s (1996) discussion, it is normal for women who undergo
menstrual cycle to have changes in their paired soft tissue traits, such as breast size and
digit length. They have observed women who undergo normal menstrual cycle each
month using a trans-abdominal real-tie ultrasonography, and that results show that there
is an increase in size for the smaller traits, while there is a decrease in size for the bigger
traits, but to a lesser degree.

Progesterone and metabolites Pregnanediol & Pregnanetriol

Low ovulation percentage is directly affiliated with unelevated levels of

progesterone in women with regular menstrual cycles aged 20-40 years experiencing
infertility. (Sanchez, ex`t al., 2016). In a case-control study, 302 selected patients were
assigned into 2 groups according to ovulatory state (i.e., Group 1 anovulatory and Group
II ovulatory). They have undergone intravaginal US and garnered comparable statistics
accentuating the significant association between the two variables. Shown in their figures
are the inverse proportion of LUF (Luteinizing unruptured follicle) in the patient’s cycle
and their P4 (Progesterone) level, a probable cause of impotency.

According to Blackwell, Vigil, Cooke, d’Arcangues and Brown (2013), Ovarian

Monitoring aids the tracking of daily excretion rates of oestrone glucuronide (EIG) and
pregnanediol glucuronide (PdG) in urine. Self-determination and re-analysis of EIG and
PdG levels on urine samples were performed and as expected, rise and falls were
observed during data analysis but exhibited significance of PdG excretion rate. An
increase or lack of, in PdG succeeding EIG peaks mark the end of the fertile window and
may suggest a luteal phase. In conclusion, PdG’s plays the vital role of determining the
start and end of ovulation, thus providing accurate results in using Ovarian Monitors.

Different Methods of Tracking Ovulation

The BodyMedia SenseWear (BMSW), a device strapped on the body, was used in
a study entitled “BMSW Testing to Track Women Ovulation” as an alternative way to
monitor ovulation of women. It charts basal temperatures, one of the more common ways
of tracking ovulation. However, results of the study showed little agreement to none even
though there was consistency of temperature readings. Other studies also shows factors
like alcohol consumption, stress levels, food eaten, and amount of work done have effect
on the results.

Another study by Houk, Kunselman and Lee (2009) made use of unstimulated LH
and Follicle-stimulating hormone to determine ovulation in young girls. Using the
Architect and Delfia assay in GnRHa stimulation were used to separate young girls with
central precocious (early development than usual) puberty and prepuberty. Earlier
versions measuring GnRHa didn't have accurate data resulting to no differences
between CCP and prepuberty. A small sample, Basal LH and FSH levels were tested in
the study. The follicle-stimulating hormone did not meet the data, while a single basal LH
was enough to determine CCP from prepuberty. Differences between both parties will
help women and doctors know who among the girls are ready to conceive a child.

Page 5 of 31
Vanillin

Vanillin, from the plant Vanilla Panifolia, but commonly obtained from eugenol, is a
flavoring agent widely used in food products as well as artificial fragrances. Two types of
vanillin are commercially available: Synthetic vanillin, which costs US$15/kg, and natural
vanillin which costs up to US$4000/kg.

A study, entitled “Vanillin Production by Phanerochaete Chrysosporium Grown on

green coconut Agro-industrial husk in solid state formation” (Barbosa, Perrone,
Vendramini, and Leite, 2008) promises a new way of producing vanillin. Using the
degradation process of white-rot fungi, which releases different chemicals, one of them
being Vanillin, the study proves to be an efficient source of vanillin. Through the Plackett-
Burman design, production of vanillin increased by 20%, an effective alternative method
of obtaining vanillin.

Testing Using Vanillin

Vanillin has a quality of working as a chemical sensor. A study which was

conducted by Herald, Gadgil, Perumal, Bean, and Wilson (2013) made use of 96-well
plate HCl-vanillin assay comparing it to the conventional methods of assay screening to
measure the tannin content of sorghum grain. It made use of 1% vanillin; mixed with 4%
HCl in methanol, and 8% concentrated HCl which is then tested to 0.2 g of Sorghum. 30
replications of both processes were used to ensure accuracy in data. The study also
made use of 3 runs of catechin at 3 different concentrations. After the repetitions and
statistical analysis, it shows that the 96-wellplate HCl-vanillin assay is an effective and
reliable method of measuring tannins, especially for large sample number.

Paper chromatography

Paper chromatography is used to separate the different components of a

substance. Various studies have used it to identify amino sugars, steroids, and others.
Paper chromatography involves the use of a filter paper and a solvent to which the paper
is suspended in. After a few hours (even days), the components shows up on the paper
as spots and these are identifiable by the different color it produces. If the spots are
colorless, a reagent is sprayed which produces a visible color. This is usually used as
chemical sensors of different studies.

Page 6 of 31
Methodology: (Provide a schematic diagram of the data collection procedures/experiment, including
materials for each process.)

Research Design

The study is of Experimental design in nature, as it seeks to determine the

effectivity of using Vanillin Perchloric Acid Paper Chromatogram in tracking ovulation of
women. It will use a purposive sampling technique in finding the respondents residing in
Metro Manila, and would only choose females between the ages of 18-40. A comparison
will be made between the Vanillin Perchloric Acid Paper Chromatogram Test, and Clinic
Test using Charting Basal Temperatures and Measuring Estrogen Levels.

Schematic Diagram:

General Procedure:

Page 7 of 31
 1. Collection and Preparations of Materials

Both Vanillin, and Chromatography paper or filter paper (depending on

availability) will be bought online. As most dealers only offer bulk orders, it is scarcely
available here in the Philippines. The Specimen cups and Perchloric Acid however, will
be bought locally here in Manila. Other needed apparatus will already be available in the
laboratory to be used.

2. Design and Construction of the Vanillin Perchloric Acid Paper Chromatogram Test

The researchers would design the schematic diagram of the kit before the actual
creation of the device. After considering all possible affecting variables, the researchers
would then construct the kit using the said materials. The Chromatography paper or the
Filter paper would be cut into small strips, as to imitate a litmus type test in detecting
fertility. Combining the Vanillin and Perchloric Acid, it would then be used in the
chromatography paper as to detect the presence of ovulation and fertility.

3. Testing the Product

To test the effectivity of the created device, the researchers would dip the test
strips in urine samples of approximately 40-100 person, and await the change of color.
The amount of the distance of change in color would determine the amount of
progesterone available. Higher progesterone levels would mean that ovulation took
place.

● Effectivity

The researchers would compare the created VPAPCT kit to conventional ways
of detecting ovulation: Charting Basal Temperatures, and Measuring Progesterone
Levels. The data that will be gathered would be subjected to statistical analysis to
determine the accuracy of each way.

● Cost Analysis

To determine if the product is cheaper to use than the clinical ways, the
researchers would conduct a cost analysis on the three said ways. The researchers
would compute for the long term expenses of the three ways, then through statistical
analysis, would determine which is the cheapest to use.

4. Collection of data and statistical analysis

The researchers would gather all data acquired from the testing, and would
subject it to statistical analysis (Tentative: One Way ANOVA). Future results would prove
that the Vanillin Perchloric Acid Paper Chromatogram Test would be the most efficient to
use, due to its cheaper cost, accurate results, and ability to be conducted at home.

Target Beneficiaries: (Identify the clienteles or recipients who will benefit from the research project. Min-Max
no of words: 20-100)

This study is concerned with women opting to measure their ovulation rate with the
aid of a self-administered device that is serviceable and relatively cheaper such as the
Vanillin Perchloric Acid Paper Chromatogram Test. It will provide a cheaper alternative to
clinical methods of detecting ovulation, while giving the same or even more accurate
result. In conjunction with this, it aims to augment the calendar method of family planning
by giving accurate determination of when women are most fertile thus give them a better
control over achieving or avoiding pregnancies.

Page 8 of 31
Expected Outputs: (Indicate the specific products, processes, and results which the project is expected to
produce and how these outputs may be used. Min-Max no. of words: 30-100)

The study aims to produce a device called “Vanillin Perchloric Acid Paper
Chromatogram test”, a self-administered and cheap device that measures progesterone
levels on urine samples, to determine fertility. Through testing of urine samples collected
from the participating women, comparison of test results from Vanillin Perchloric Acid
Paper Chromatogram Test (Henceforth: VPAPCT), Basal Body Temperature Test, and
Measurement of Estrogen Levels will show that VPAPCT is the most effective and
accurate in determining ovulation. The strip type test kit would reach a certain level of
coloration, indicating fertility; a simple process, easy to use, yet very accurate.

References: (Make a reference list using APA format 6th edition.)

● Barbosa, E. D., Perrone, D., Vendramini, A. A., & Leite, S. F. (2008). Vanillin
Production by Phanerochaete Chrysosporium Grown on green coconut Agro-
industrial husk in solid state formation. BioResources, 3(4), 1042-1050.
Retrieved May 3, 2017, from
http://ojs.cnr.ncsu.edu/index.php/BioRes/article/view/BioRes_03_4_1042_Barb
osa_PAF_Vanillin_Green_Coconut_Husk
● Blackwell, L. F., Vigil, P., Cooke, D. G., D'arcangues, C., & Brown, J. B.
(2013). Monitoring of ovarian activity by daily measurement of urinary
excretion rates of oestrone glucuronide and pregnanediol glucuronide using
the Ovarian Monitor, Part III: Variability of normal menstrual cycle profiles.
Human Reproduction,28(12), 3306-3315. doi:10.1093/humrep/det389
● Boyes, W. (2010). 22. Chemical Analysis: Introduction. In Instrumentation
Reference Book (4th Edition) (pp. 328–329). Elsevier. Retrieved from
http://app.knovel.com/hotlink/pdf/id:kt007DOK74/instrumentation-
reference/chemical-analysis-introduction
● Ecochard, R., Leiva, R., Bouchard, T., Boehringer, H., Direito, A., Mariani, A.,
& Fehring, R. (2013). Use of urinary pregnanediol 3-glucuronide to confirm
ovulation. Steroids, 78(10), 1035–1040.
https://doi.org/10.1016/j.steroids.2013.06.006
● Herald, T. J., Gadgil, P., Perumal, R., Bean, S. R., & Wilson, J. D. (2013).
High-throughput micro-plate HCl–vanillin assay for screening tannin content in
sorghum grain†. J Sci Food Agriculture, 94, 2133-2136. doi:10.1002/jsfa.6538
● Hsiu-Wei Su, Yi, Y.-C., Wei, T.-Y., Chang, T.-C., & Cheng, C.-M. (2017).
Detection of Ovulation, a Review of Currently Available Methods.
Bioengineering & Translational Medicine. https://doi.org/10.1002/btm2.10058
● Sanchez, E. G., Giviziez, C. R., Sanchez, H. M., Agostinho, P. L., Barros, P.
S., & Approbato, M. S. (2016). Low progesterone levels and ovulation by
ultrasound assessment in infertile patients. JBRA Assisted Reproduction,20(1),
13-16. doi:10.5935/1518-0557.20160004
● Wilson, I. D., & Poole, C. F. (2009). Paper Chromatography. In Handbook of
Methods and Instrumentation in Separation Science, Volume 1 (pp. 621–628).
Elsevier. Retrieved from
http://app.knovel.com/hotlink/pdf/id:kt00BYY541/handbook-methods-
instrumentation/paper-chromatography
● Wark, J. D., Henningham, L., Gorelik, A., Jayasinghe, Y., Hartley, S., &
Garland, S. M. (2015). Basal temperature measurement using a multi-sensor
armband in Australian young women: A comparative observational study.

Page 9 of 31
 JMIR Mhealth Uhealth, 3(4), 1-25.
DOI:https://dx.doi.org/10.2196%2Fmhealth.4263
● Houk, C. P., Kunselman, A. R., & Lee, P. A. (2009). Adequacy of a single
unstimulated luteinizing hormone level to diagnose central precocious puberty
in girls. Pediatrics, 123(6), 1-7. DOI:10.1542/peds.2008-1180

Note: Table may be adjusted to accommodate entries.

BUDGETARY REQUIREMENTS

ITEMS AMOUNT (in PHP) REMARKS

A. Materials

- Paper 120 Pesos - 40pcs

- Paper Filter 1000 Pesos -
- Chromatography Paper 100pcs

- Vanillin 175 Pesos - 10g

- Perchloric Acid 1470 Pesos - 500ml

- Specimen Bottle 30 Pesos/pc

- UST Hospital
- C. Miranda

B. Services (Lab testing fees, etc.)

- Best Diagnostic Corp. 800 Pesos

C. Others (Please specify)

Page 10 of 31
 - Travel Finance (Initial) 1000 Pesos

- Total Shipment Approx. 1500 Pesos

TOTAL

Note: Table may be adjusted to accommodate entries.

WORK PLAN

ACTIVITIES IMPLEMENTATION OUTPUT

DATE

Canvassing of Materials August 7 - 18 Complete materials needed

Test Kit Making (with packaging) August 21 - Finish kit of the Vanillin
September 18 Perchloric Acid Paper
Chromatogram Test

Interview with an Ob-Gyne (pre) September 17 Opinions of the Ob-gyne

Page 11 of 31
Experiment Proper (1st month) September 17 - Results of Tests of the VPAPCT,
October 14 and clinical tests

Experiment Proper (2nd month) October 14 - Results of Tests of the VPAPCT,

November 11 and clinical tests

Interview with an Ob-Gyne (post) December 4 Suggestions and

recommendations of the Ob-gyne

Submission of Research Paper Research paper

Note: Table may be adjusted to accommodate entries.

LOUIE B. DASAS, LPT, MSc

Research Teacher
Signature over Printed Name

Page 12 of 31
October 1, 2017
Dra. Sarah Cajusay-Velasco, OB/GYN
St. Margaret Clinic
142 Poblacion Bocaue, Bulacan

RE: Permission to Conduct Study

Greetings!
We are writing to humbly request for your permission to conduct our research study in your
institution. We are grade 12 students from the Health Allied strand of the University of Santo
Tomas.
This said experiment will mainly focus on predicting the ovulation cycle of women through
the use of Vanillin Perchloric Acid Paper Chromatogram Testing and we sincerely hope that
your institution will allow us to collect the needed fluid samples (i.e. urine) of your selected
patients in order to push through with the research. We also humbly ask for your help regarding
the professional assistance of an obstetrician gynecologist that can aid us to meet the criteria and
the expected outcome of our experiment. Due to the sensitive nature of the study, participating
patients will be given a consent form to sign and returned to the primary researchers at the
beginning of the sampling process. The results will be pooled for the thesis project and we assure
you the anonymity and confidentiality of each and every participant involved in the study.
Should this be published, only the pooled results will be documented and no cost of all forms
shall be shouldered by you nor the institution you belong in.
Your approval to this study will be greatly appreciated as we look forward to a healthy
collaboration with you and your institution as we go through the journey of the completion of
this study. Should you have any concerns or questions regarding our proposal, kindly e-mail us
at johnpatrick.lui.shs@ust.edu.ph or send a message to 09178814754.
We hope to hear from you soon.

Sincerely,
12HA-3 Group 9
Batac, FranchesCa C.
Cuevas, Ma. Regina Alexa E.
Lui, John Patrick C.
Nuñez, Keziah Mae T.D.
Reyes, Mikaella Louisse D.

Page 13 of 31
 CONSENT FORM FOR PARTICIPANTS IN THE STUDY

RESEARCH TITLE: EFFECTIVITY OF VANILLIN PERCHLORIC ACID PAPER

CHROMATOGRAM KIT IN DETERMINING OVULATION OF WOMEN AGES 18 TO 25.

RESEARCHERS: BATAC, Franchesca, CUEVAS, Ma. Regina Alexa, LUI, John Patrick,
NUÑEZ, Keziah Mae, REYES, Mikaella

I have been given information about EFFECTIVITY OF VANILLIN PERCHLORIC

ACID PAPER CHROMATOGRAM KIT IN DETERMINING OVULATION OF WOMEN
AGES 18 TO 25 and discussed the research project with Ms. Batac, Ms. Cuevas, Mr. Lui, Ms.
Nuñez, and Ms. Reyes, who are conducting this research as part of their study in their Practical
Research II, supervised by Mr. Clairefrancis De Guzman in the Department of Research Center
for Natural and Applied Sciences at the University of Santo Tomas.

I have been advised of the potential risks and burdens associated with this research, which
include the confidentiality of my participation and the sample acquired from me, and have had an
opportunity to ask any of the aforementioned researchers any questions I may have about the
research and my participation. I understand that my participation in this research is voluntary, I
am free to refuse to participate and I am free to withdraw from the research at any time.

If I have any enquiries about the research, I can contact John Patrick Lui at 09178814754
or if I have any concerns or complaints regarding the way the research is or has been conducted,
I can contact the SHS office at the University of Santo Tomas on +63-2-406-1611 or email rso-
socialmedia@ust.edu.ph By signing below I am indicating my consent to

(please tick):
Acquire a urine sample from me willingly.

Actively and truthfully answer the researcher’s questions.

Allow to be monitored and observed.

I understand that the data collected from my participation will be used for purpose (eg thesis,
journal publication, etc), and I consent for it to be used in that manner.

Signed Date (mm/dd/yy)

Name in Printed Form

Page 14 of 31
 THE RESEARCH METHODS

The experiment to be conducted is about determining the ovulation cycle of women ages

18 to 25 using a device made of Vanillin perchloric acid. It will be a chromatogram type of test

wherein a series of steps must be followed to yield the result the researchers are trying to

obtain. With this experiment, information from different journals and other reference materials

are cited.

The first main point of the methodology is the reagents and chemicals to be used which

include concentrations of Vanillin, the primary component of the extract of vanilla bean. It has

a pleasant odor and taste, with a color of powdery white or a yellowish tint. It can be used as an

alternative to vanilla in food and beverages, and is also used in pharmaceuticals. But it has its

danger hazard which may be cause allergic reactions and eye irritation; b) Toluene-p-sulphonic

acid is an organic compound that is white in color. It is soluble in polar solvents. This acid is

used in different areas such as in medications, cleaning, and furnishing, as an additive, and

buildings or constructions. It has its warning hazard wherein it can lead to eye and respiratory

damage. Inhalation and ingestion should be avoided; c) Perchloric acid, a colorless acid with a

pungent odor has a danger hazard and should be avoided in the means of inhalation and

ingestion. It is used in laboratories as an oxidizing or reducing agent that can cause fire,

explosion, corrosion, and severe damage to the body if not handled properly; and d) water,

which is used as a solvent for many substances. It has no color and taste. It is essential in

human, animal, and plant life, and is used in a variety of ways. It can also have its hazard if not

handled properly.

Page 15 of 31
 The second main point is the extraction process. This is where the reagent to be used in

the study will be made using the chemicals and substances listed above. In different

concentrations they will be combined together and a process of heating, drying, placing spots

of the urine on the chromatogram paper will be done.

The third main point includes the instruments and the interpretation of data. The equipments

will be used during the extraction process and to hold the substances are beakers, clips and

sticks, pipettes, and heaters. Wearing the personal protective equipment (PPE) which includes

the laboratory gown, goggles, mask, and gloves is the most important equipment to be used to

protect the researchers from any hazard. During data interpretation, change in color of the

chromatogram test, grouping the tests based on the age bracket, using Person’s Chi-squared

test and Cost-Effectiveness Analysis (CEA) following that of Chris Edwards (2011) will be

performed.

There are two formal sub-sections (3.1 and 3.2) which are;

3.1 Methods and Materials

3.1.1 Standards, Reagents, and Chemicals

A filter paper will be used for the detection of Pregnanetriol, and the most suitable

reagent based on J.D. Few’s 1961 study on Pregnanetriol and other steroids detection through

chromatography will be formulated. The reagent to be used for the detection of steroids—

particularly Pregnanetriol—will contain the following:

 1 g of Vanillin

 10 g of toluene-p-sulphonic acid

Page 16 of 31
  15 mL of 60% Perchloric Acid

 Water (to 100mL)

(A 10% v/v concentration of Perchloric Acid is preferred in terms of color-yield accuracy neither

without damaging the paper nor without the disadvantages associated with an increase of the

Perchloric Acid concentration.)

Color Forming Reagents

 Liquid form: 1 percent aldehyde, 10 percent perchloric acid and toluene-p-sulphuric

acid/orthophosphoric acid

 Aldehydes soluble in water: 20 percent acetone included 1 g vanillin, 10 g toluene-p-

sulphuric acid, and 15 ml of 60 percent perchloric acid plus water to 100 ml

3.1.2 Extraction Procedure

First, make a reagent A by combining 5 parts of iso-octane, 15 parts of toluene, 16 parts of

methanol, 4 parts water, or, 8 parts of iso-octane, 12 parts of toluene, 16 parts of methanol,

and 4 parts of water. Dip a dry, filter paper on the reagent A, and afterwards apply spots of

urine approximately 5 micrograms per sq/cm on filter paper.

Proceed to making reagent B; the Vanillin concentration consisting of 1 percent aldehyde,

10 percent perchloric acid, and toluene-p-sulphuric acid or orthophosphoric acid. Dip the

treated dried paper (from reagent A) into reagent B and remove the excess solution through

blotting using a spongy, unsized paper for absorbing ink. Dry the paper with solution on warm

Page 17 of 31
air, with temperatures ranging from 75 𝐶 0 to 85 𝐶 0 , using laboratory air blower or electric

convection heater.

Make sure temperature does not exceed 100 𝐶 0 as the steroid sample will be destroyed).

Observe: as a result, the Vanillin reagent will produce yellow spots (from steroids);

appearing on a yellow background, which will fade rapidly. Spots will fade after 30 minutes but

reheating to 85 𝐶 0 will make it reappear.

3.2 Analysis

3.2.1 Instrumental Analysis

 Beakers/Glass containers

o Where mixtures shall be mixed to observe proper chemical handling and safety

measures.

 Binder Clip and Stick

o Something to hold the chromatography paper while being dipped into the

solution.

 Glass Capillary/ Pipette

o This will load the sample (urine) onto the paper.

 Electric Convention Heater / Laboratory Air Blower

o This instrument will be used to dry the papers through steaming in warm air

ranging from 750 to 850C.

 PPE (Personal Protective Equipment)

o Face Mask

Page 18 of 31
 o Gloves

o Laboratory Gown

o Goggles

o Wear this to ensure safety of the members while performing the procedure.

3.2.2 Data Interpretation and Calculations

The results of the test kits will be checked based on the color change after being dipped in

the urine for 2 to 3 minutes. If a change of color is evident and detected, this will indicate the

effectiveness of the kit in detecting progesterone steroid- pregnanetriol. The same urine

samples will also be subjected to laboratory methods of detecting ovulation. The result will

then be used as basis of comparison on the effectiveness of the created kit.

The test kit results will be grouped according to the age of the participant in order to

categorize the results closely. This will give the researchers 8 groups for the statistical analysis

of data.

Pearson’s Chi-squared test will be used at p<0.05 error, to compare the number of VPA

kits that showed ovulation versus the number of results of laboratory testing. A comparison of

frequency distribution of the effectiveness of the VPA test kit will be performed using IBM

software - SPSS, with the laboratory results acting as the theoretical distribution of determining

ovulation. A 5% error (0.05) will be used to ensure accuracy of statistical computations. If

results will have error level below p<0.05, this will infer that the VPA test kit is relatively

effective as the laboratory testing.

Page 19 of 31
 A Cost-Effectiveness Analysis (CEA) will also be performed, patterned to the Cost-

Effectiveness Analysis in Practice vol. 10 by Chris Edwards (2011). This will help determine if

the VPA test kit is much cheaper compared to laboratory methods.

Page 20 of 31
 BIBLIOGRAPHY

Barbosa, E. D., Perrone, D., Vendramini, A. A. & Leite, S. F. (2008). Vanillin production by

Phanerochaete Chrysosporium grown on green coconut agro-industrial husk in solid

state formation. BioResources, 3(4), 1042-1050. Edwards, C. (2011). Cost-effectiveness

analysis in practice - WHO. Retrieved October 5, 2017 from

http://www.bing.com/cr?IG=17FB3C5FC7A84BC59CDDF7986F05E24E&CID=0D06F4B18

E9C6FF82C91FFA38F9A6EDE&rd=1&h=m-

mOAV2VfT8hvSqJ76rkD7MQHGXgQ5QEOiAeyqROQjM&v=1&r=http%3a%2f%2fwww.w

ho.int%2fwater_sanitation_health%2fpublications%2f2011%2fch10.pdf&p=DevEx,5065.

Blackwell, L. F., Vigil, P., Cooke, D. G., D'arcangues, C. & Brown, J. B. (2013). Monitoring of

ovarian activity by daily measurement of urinary excretion rates of oestrone glucuronide

and pregnanediol glucuronide using the Ovarian Monitor, Part III: Variability of normal

menstrual cycle profiles. Human Reproduction, 28(12), 3306-3315.

doi:10.1093/humrep/det389

Boyes, W. (2010). 22. Chemical analysis: Introduction (4th ed). Amsterdam, NE: Elsevier.

Ecochard, R., Leiva, R., Bouchard, T., Boehringer, H., Direito, A., Mariani, A. & Fehring, R. (2013).

Use of urinary pregnanediol 3-glucuronide to confirm ovulation. Steroids, 78(10), 1035–

1040. https://doi.org/10.1016/j.steroids.2013.06.006

Few, J. D. (1961). A Vanillin - perchloric acid reagent for detecting Pregnanetriol and related

compounds on paper chromatograms. Department of Chemical Pathology, 86, 636-640.

Page 21 of 31
Generalic, E. (2017). Filter paper. Croatian-English Chemistry Dictionary & Glossary. Retrieved

October 5, 2017 from https://glossary.periodni.com/glossary.php?en=filter paper

Green B., Tarte, J., & Waller, M. (2017). What is cost analysis? Retrieved October 5, 2017 from

http://www.homevisitcosts.com/what-is-cost-analysis.php

Group, E. (2014). What is vanillin? Retrieved October 5, 2017 from

https://www.globalhealingcenter.com/natural-health/vanillin/

Herald, T. J., Gadgil, P., Perumal, R., Bean, S. R. & Wilson, J. D. (2013). High-throughput micro-

plate HCl–vanillin assay for screening tannin content in sorghum grain†. J Sci Food

Agriculture, 94, 2133-2136. doi:10.1002/jsfa.6538

Hsiu-Wei Su, Yi, Y.-C., Wei, T.-Y., Chang, T.-C. & Cheng, C.-M. (2017). Detection of ovulation, a

review of currently available methods. Bioengineering & Translational Medicine.

https://doi.org/10.1002/btm2.10058

Houk, C. P., Kunselman, A. R., & Lee, P. A. (2009). Adequacy of a single unstimulated luteinizing

hormone level to diagnose central precocious puberty in girls. Pediatrics, 123(6), 1-7.

DOI:10.1542/peds.2008-1180

IBM. (2016). IBM SPSS Software. Retrieved October 5, 2017, from

https://www.ibm.com/analytics/us/en/technology/spss/

Irimia, R. E. & Gottschling, M. (2016). Taxonomic revision of Rochefortia Sw. (Ehretiaceae,

Boraginales). Biodiversity Data Journal 4: e7720. https://doi.org/10.3897/BDJ.4.e7720

Omnisurge Medical Supplies. (n.d.). Urine sample bottle | specimen jar. Retrieved October 5,

2017 from http://omnisurge.co.za/shop/urine-sample-bottle-specimen-jar/

Page 22 of 31
One Green Planet. (2014). Vanilla versus vanillin: what you need to know. Retrieved October 5,

2017 from http://www.onegreenplanet.org/natural-health/vanilla-versus-vanillin-what-

you-need-to-know//

PubChem. (n.d.). PERCHLORIC ACID. Retrieved October 5, 2017 from

https://pubchem.ncbi.nlm.nih.gov/compound/perchloric_acid#section=Top

PubChem. (n.d.). P-TOLUENESULFONIC ACID. Retrieved October 6, 2017 from

https://pubchem.ncbi.nlm.nih.gov/compound/p-toluenesulfonic_acid#section=Top

PubChem. (n.d.). Vanillin. Retrieved October 6, 2017 from

https://pubchem.ncbi.nlm.nih.gov/compound/vanillin#section=Top

PubChem. (n.d.). Water. Retrieved October 6, 2017 from

https://pubchem.ncbi.nlm.nih.gov/compound/water#section=Top

Quirky Science. (2017). Vanilla and vanillin: What's the difference? Retrieved October 5, 2017

from http://www.quirkyscience.com/vanilla-and-vanillin/

Sanchez, E. G., Giviziez, C. R., Sanchez, H. M., Agostinho, P. L., Barros, P. S. & Approbato, M. S.

(2016). Low progesterone levels and ovulation by ultrasound assessment in infertile

patients. JBRA Assisted Reproduction, 20(1), 13-16. doi:10.5935/1518-0557.20160004

What are the apparatus used for paper chromatography? (2017). Retrieved October 5,

2017 from https://www.quora.com/What-are-the-apparatus-used-for-paper-

chromatography

VWR. (n.d.) Urine specimen bottle. Retrieved October 5, 2017 from

https://uk.vwr.com/store/product/545363/urine-specimen-bottle.

Wark, J. D., Henningham, L., Gorelik, A., Jayasinghe, Y., Hartley, S. & Garland, S. M. (2015).

Page 23 of 31
 Basal temperature measurement using a multi-sensor armband in Australian young

women: A comparative observational study. JMIR Mhealth Uhealth, 3(4), 1-25.

DOI:https://dx.doi.org/10.2196%2Fmhealth.4263

Wilson, I. D. & Poole, C. F. (2009). Paper chromatography. Amsteram, NE: Elsevier.

Page 24 of 31
 Timetable for Research

Research Objectives 2017 2018

Aug Sep Oct Nov Dec Jan Feb Mar Apr May
1. Gathering of materials
2. Making of the kit.

3. Identifying samples.

4. Testing of the kit.

5. Comparison to other
methods of determining
ovulation.
6. Collection of data results.
7. Treatment and analysis;
statistics of data.
8. Defense.

Page 25 of 31
 Budgetary Requirements

SOURCES OF EXPENSES Quantity Amount

(PhP)
Vanillin Powder 10g 200
Perchloric Acid 500mL 1,500
Filter Paper 1 sheet 35
Specimen Cup 1 piece 30

Total PhP1, 765

Page 26 of 31
FRANCHESCA C. BATAC

#2409 Josefa St., Josefa Subdivision,

Balibago, Angeles City, Pampanga

chescacunan@yahoo.com

09562636923

Franchesca C. Batac graduated junior high school at Holy Angel University as a consistent

academic achiever, where she has taken up research subjects and proposals as part of the

curriculum. She was part of the school choir from 2013 to 2015 as the president. Currently,

she is enrolled at University of Santo Tomas as a grade 12 student under the Health Allied

strand.

Page 27 of 31
MA. REGINA ALEXA E. CUEVAS

814 J.P. Rizal St. Nangka Marikina City, NCR

alexaecvs@gmail.com

09176333678

Ma. Regina Alexa E. Cuevas completed junior high school at St. Scholastica’s Academy

Marikina (SSAM) in 2016. During her time in the aforementioned school, she became a merit

awardee when she was in Grade 8 and 9. Moreover, she held the position of grade level

representative (2013-2014) and auditor (2015-2016) in her organization, Student Organization

for Social Actions. When she was in 10th grade, she became a student teacher for Grade 10

students in their Guidance subject. Currently, she is enrolled at the Pontifical and Royal

University of Santo Tomas as a Grade 12 student under the Health Allied Strand. She is also an

auditor in her class and current organization, the Crisis Control Council. Additionally, she is a

volunteer for the Health Allied Strand Society.

Page 28 of 31
KEZIAH MAE T.D. NUÑEZ

Blk 11, lot 32, Sunshine Fiesta Subdivision,

Tatala, Binangonan, Rizal.

839 P.Campa ST. Sampaloc, Manila. Brgy 396, Loyola and Espanya.

keziahmae.nunez.shs@ust.edu.ph

09338773727

Keziah Mae Tiu Dy Nuñez graduated from St. Jerome’s Academy in 2016 in third honors.

She has won several local quiz bees held within her former school and had been an officer in

several monthly activities, from being the SC 4th year representative, to being the English club

month vice president. She is also a former member of the lectors and commentators parish

organization and a catechist in her former school; teaching children from grade 1 to 2 in a span

of 2 years. She is currently a grade 12 student at UST Senior High School under the Health Allied

Strand.

Page 29 of 31
MIKAELLA LOUISSE D. REYES

7B Del Rosario St. Dampalit, Malabon

mikaellalouissereyes@gmail.com

09055498193/7095824

Mikaella Louisse D. Reyes graduated at St. Therese of the Child Jesus Academy in the year

2016. She also received a loyalty award and an academic excellence award for ranking 9 th

among their batch. She has hosted several Investigatory Project Contests for 4 years in her

Alma Mater and is currently studying at the University of Santo Tomas Senior High school under

the Health Allied Strand.

Page 30 of 31
JOHN PATRICK C. LUI

Unit 23, 28th Flr. Tower 1, Amaia Skies Avenida,

Doroteo Jose St., Sta. Cruz, Manila

johnpatrick.lui.shs@ust.edu.ph

09178814754

John Patrick C. Lui graduated junior high school from Philippine Yuh Chiau School in

2016 as the first honor, and is now a Santo Tomas scholar of the University of Santo Tomas. He

is currently a grade 12 student in the health allied strand.

He has been trained in the field of research since 2013. He started joining research

competitions in 2014, and has become a champion in Division Science and Technology fair

2014-2015, first runner up Division Science and Technology Fair 2015-2016, and first runner up

in Regional Science and Technology Fair 2014-2015. He has been rewarded as the best

researcher in his previous school for 2 consecutive years, using the research he created

entitled: “Harnessing Human Energy through the use of Piezoelectric Transducers” which also

garnered him the said awards in the Science and Technology Fair competitions. He has also

joined a lot of science quiz bees like the Philippine Nuclear Science Quiz and MATHSCIAKA, and

received awards as 2nd place winner in 2014 Northern Luzon Science & Technology Fair, which

gave him broad information regarding current science and technology developments.

His experience in his family business and his experience in presenting his previous

researches also has been a beneficial tool by giving him the confidence in presenting in front of

audiences and judges.

Page 31 of 31