software was used and the chromatog

software was used and the chromatograms were monitored at
285 nm, 350 and 450 nm.
The identification was made by comparison of their chromatographic and UV–vis spectroscopic characteristics with those of
standards.
The quantification was carried out by external calibration from
the areas of the chromatographic peaks obtained by DAD detection at the following wavelengths: 285 nm for phytoene and TOCS,
350 nm for phytofluene and 450 nm for CARS and CHLS.
2.5. Method validation
Linearity of the method was evaluated by considering of the
detector response (area units) to different amounts (g) of CARS,
CHLS and TOCS by means of linear regression. In order to satisfy
basic requirements such as homoscedasticity and linearity, the Ftest and the residual plot were performed at the 95% significance
level [35,36].
Stock solutions of the individual compounds were prepared separately. The concentrations of the working standards
were determined spectrophotometrically using published molar
absortivity values (εmol) as indicated in Supplementary material.
The desired concentration range for the preparation of standard
curves was obtained by serial dilution. The calibration curves
were constructed by plotting the response area against the corresponding concentration injected. The limits of detection (LOD) and
quantification (LOQ) were calculated from the calibration curves,
using the Microcal Origin ver. 3.5 software (OriginLab Corporation,
Northampton, MA, USA). The LOD were calculated as three times
the relative standard deviation of the analytical blank values calculated from the calibration curve. The LOQ were calculated as ten
times the relative standard deviation of the analytical blank values
calculated from the calibration curve.
To evaluate the precision and recovery of the methods, four samples were selected considering its composition; chard as a source
of both CHLS and CARS (mainly 9

-cis-neoxanthin + violaxanthin,
lutein, and -carotene), carrot as a source of carotenes (mainly, -and -carotene) and TOCS, red peach as a source of -cryptoxanthin
and grapefruit as a good source of lycopene, -carotene and colorless carotenoids (phytoene and phytofluene).
The within-laboratory repeatability (within-day precision) was
developed according to UNE 82009 standards [37]. It was ascertained by analyzing the CARS, TOCS and CHLS content in samples,
under the same analytical conditions. Three replicates from each
sample were extracted and all the samples and standards were
injected three times.
Within-laboratory reproducibility (day-to-day precision) was
assessed by extracting and analyzing the CARS at 2-day intervals
during 3 days.
A recovery study was performed to validate the accuracy of the
developed method. It was expressed as the percentage recovery,
which was calculated as:
%Re =
observed concentration − theoretical concentration
theoretical concentration
× 100
The accuracy of the method was evaluated spiking with known
quantities of different standards (trans--apo-8

-carotenal (APO),
chlorophyll a (CHLA), -carotene (BCAR) and -tocopherol (ATOC).
The spiked samples were then extracted and analyzed with the
proposed RRLC method.
To evaluate the recovery of carotenoids, the samples of chard,
carrot and grapefruit were spiked with 100 l of trans--apo-8

-carotenal (dissolved in acetone at a concentration of 432 mg/l) and
then extracted by the methodology described above (Section 2.3).
Trans--apo-8

-carotenal is a synthetic carotenoid that has been
traditionally used as internal standard in some laboratories [38].
Likewise, to evaluate the recovery of CHLS and TOCS, samples
of banana were spiked with 20 l of CHLA (dissolved in acetone at
a concentration of 910 mg/l) and with 50 l of ATOC (dissolved in
acetone at a concentration of 1870 mg/l) and then extracted by the
methodology described above (Section 2.3).
On the other hand, the effect of saponification in the recovery of
CARS and TOCS was evaluated. For this purpose, the same samples
of banana were spiked with 200 l of BCAR (dissolved in acetone
at a concentration of 540 mg/l) and with 50 l of ATOC (dissolved
in acetone at a concentration of 1870 mg/l). Finally, these samples
were extracted and saponified by the methodology described above
(Section 2.3).
To reduce to the minimum sources of errors, recoveries were
evaluated in banana because this sample had the least complex
carotenoid profile among the products analyzed.
3. Results and discussion
Different assays were carried out to optimize the chromatographic conditions in order to obtain a suitable separation of the
isoprenoids in the extracts. For this, a mixture of standard was
used; Figs. 1 and 2 show the chromatograms of the standard mixture using the optimized conditions, detailed in Section 2.4. The
developed method allows the separation seventeen compounds in
the fruit and vegetables in a 12-min run. Specifically a mixture of
nine CARS (violaxanthin, zeaxanthin, zeinoxanthin -cryptoxathin,
-carotene, -carotene, lycopene, phytoene, phytofluene), four
tocopherols and four chlorophylls and derivatives (chlorophylls
and pheophytins) was separated.
3.1. Analytical characteristics
3.1.1. Linearity and limits of detection and quantification
The validating parameters of each calibration curve (slope,
intercept, coefficients of determination, LOD and LOQ) are shown
in Table 1. The linear calibration range used was tested for
homoscedasticity to confirm the application of the linear leastsquares method (constant variance).
All curves showed good linearity (R
2
> 0.996) in the range of concentrations studied. As regards to carotenoids, LODs ranged from
0.001 g in phytofluene to 0.070 g in lycopene. These limits are
lower than those recently reported [39].
Concerning the tocopherols, LODs ranged between 0.007 g for
-tocopherol and 0.067 g for -tocopherol, while those of chlorophylls ranged from 0.004 g (for pheophytin b) to 0.080 g (for
chlorophyll b).
Depending on the compound, LOQs ranged from 0.002 g to
0.268 g (for phytofluene and chlorophyll b, respectively). The
results obtained for the quantitation limits show that the proposed
method is sensitive enough for the determination of dietary isoprenoid compounds of interest.
3.2. Precision and accuracy
The repeatability and reproducibility were evaluated by considering the relative standard deviation (Table 2). Concerning
repeatability, the RSD values of the method for unsaponified
samples were under 7.2%. The highest values corresponded to -carotene (7.18%) and the lowest ones to violaxanthin (0.58%). Both
values were obtained in the analyses of carrot. The highest RSD
observed in the reproducibility corresponded to -tocopherol in
the carrot sample (11.87%) and the lowest ones to lutein (4.66%)
in the chard sample. Nonetheless, most of the RSD values obtained
were below 12%, which confirmed the high reproducibility of the
method

Hasil (Bahasa Indonesia) 1:

perangkat lunak ini digunakan dan chromatograms sedang dipantau di285 nm, 350 dan 450 nm.Identifikasi dibuat oleh perbandingan mereka kromatografi dan UV-vis karakteristik spektroskopi dengan orang-orangstandar.Kuantifikasi dilaksanakan oleh kalibrasi eksternal daridaerah puncak kromatografi diperoleh oleh ayah deteksi panjang gelombang berikut: 285 nm untuk phytoene dan TOCS,350 nm untuk phytofluene dan 450 nm untuk mobil dan CHLS.2.5. metode validasiLinearitas dari metode dievaluasi dengan mempertimbangkan daridetektor respons (daerah unit) untuk jumlah yang berbeda (g) Mobil,CHLS dan TOCS dengan menggunakan regresi linear. Dalam rangka memenuhipersyaratan dasar seperti homoscedasticity dan linearitas, Ftest dan plot sisa dilakukan pada signifikans 95%tingkat [35, 36].Saham solusi senyawa individu telah dipersiapkan secara terpisah. Konsentrasi standar kerjabertekad spectrophotometrically menggunakan diterbitkan molarabsortivity nilai-nilai (εmol) seperti ditunjukkan dalam bahan tambahan.Kisaran konsentrasi yang diinginkan untuk penyusunan standarkurva diperoleh oleh serial pengenceran. Kurva kalibrasidibangun oleh merencanakan daerah respon terhadap konsentrasi sesuai disuntikkan. Batas-batas deteksi (LOD) dankuantifikasi (LOQ) yang dihitung dari kurva kalibrasi,menggunakan perangkat lunak ver. 3.5 Microcal asal (OriginLab Corporation,Northampton, MA, USA). LOD dihitung sebagai tiga kalideviasi standar relatif nilai-nilai kosong analitis dihitung dari kurva kalibrasi. LOQ dihitung sebagai sepuluhkali deviasi standar relatif nilai-nilai kosong analitisdihitung dari kurva kalibrasi.Untuk mengevaluasi presisi dan pemulihan dari metode, empat sampel dipilih mengingat komposisi; Chard sebagai sumberCHLS dan mobil (terutama 9-cis-neoxanthin + violaxanthin,lutein dan - karoten), wortel sebagai sumber karotin (terutama,- dan - karoten) dan TOCS, peach merah sebagai sumber - cryptoxanthindan jeruk sebagai sumber baik lycopene, - karoten dan karotenoid berwarna (phytoene dan phytofluene).Pengulangan dalam laboratorium (presisi dalam hari) adalahdikembangkan sesuai UNE 82009 standar [37]. Itu adalah dipastikan dengan menganalisis Mobil, TOCS dan CHLS konten dalam sampel,di bawah kondisi analitis yang sama. Tiga Ulangan dari masing-masingsampel diambil dan semua sampel dan standardisuntikkan tiga kali.Dalam laboratorium reproduktibilitas (sehari-hari presisi) adalahdinilai oleh ekstraksi dan menganalisis mobil pada interval 2-hariselama 3 hari.Pemulihan studi ini dilakukan untuk memvalidasi keakuratanmetode yang dikembangkan. Dinyatakan sebagai persentase pemulihan,yang dihitung sebagai:% Kembali =konsentrasi diamati − teoritis konsentrasikonsentrasi teoritis× 100Keakuratan metode dievaluasi spiking dengan dikenaljumlah standar yang berbeda (trans-- apo-8-carotenal (APO),klorofil a (CHLA), - karoten (BCAR) dan - tokoferol (ATOC).Sampel berduri kemudian diekstraksi dan dianalisis denganmetode RRLC yang diusulkan.Untuk mengevaluasi pemulihan karotenoid, sampel chard,wortel dan grapefruit berduri dengan 100 l trans-- apo-8-carotenal (dilarutkan dalam aseton pada konsentrasi 432 mg/l) dankemudian diekstraksi dengan metodologi yang dijelaskan di atas (Bagian 2.3).Trans-- apo-8-carotenal adalah karotenoid sintetis yang telahsecara tradisional digunakan sebagai standar internal di beberapa laboratorium [38].Demikian juga, untuk mengevaluasi pemulihan CHLS dan TOCS, sampelpisang yang berduri dengan 20 l CHLA (dilarutkan dalam aseton dikonsentrasi 910 mg/l) dan dengan 50 l ATOC (dilarutkan dalamaseton pada konsentrasi 1870 mg/l) dan kemudian diambil olehmetodologi yang dijelaskan di atas (Bagian 2.3).Di sisi lain, efek saponifikasi dalam pemulihanMOBIL dan TOCS dievaluasi. Untuk tujuan ini, sampel samapisang yang berduri dengan 200 l BCAR (dilarutkan dalam asetonpada konsentrasi 540 mg/l) dan dengan 50 l ATOC (dibubarkandi aseton pada konsentrasi 1870 mg/l). Akhirnya, contoh-contoh inidiekstraksi dan saponified oleh metodologi yang dijelaskan di atas(Bagian 2.3).Untuk mengurangi ke sumber-sumber minimal kesalahan, pemulihan yangdievaluasi dalam pisang karena hal ini sampel yang paling kompleksProfil karotenoid diantara produk dianalisis.3. hasil dan diskusiBerbagai tes dilakukan untuk mengoptimalkan kromatografi kondisi untuk mendapatkan cocok pemisahanisoprenoids di ekstrak. Untuk ini, campuran standar adalahdigunakan; Rajah-rajah 1 dan 2 menunjukkan chromatograms campuran standar menggunakan kondisi optimal, rinci di bagian 2.4. Themengembangkan metode memungkinkan pemisahan tujuh belas senyawa dalambuah dan sayuran dalam 12 menit berjalan. Khusus campuransembilan mobil (violaxanthin, zeaxanthin, zeinoxanthin - cryptoxathin,-karoten, - karoten, lycopene, phytoene, phytofluene), empattokoferol dan empat chlorophylls dan derivatif (chlorophyllsdan pheophytins) terpisah.3.1. analitis karakteristik3.1.1. linearitas dan batas-batas Deteksi dan kuantifikasiParameter memvalidasi setiap kurva kalibrasi (kemiringan,««««mencegat, Koefisien tekad, LOD dan LOQ) yang akan ditampilkandalam tabel 1. Kisaran kalibrasi linier digunakan diuji untukhomoscedasticity untuk mengkonfirmasi penerapan metode linear leastsquares (konstan varians).Semua kurva menunjukkan baik linearitas (R2> 0.996) dalam kisaran konsentrasi belajar. Sebagai hal karotenoid, LODs berkisar0.001 g di phytofluene untuk 0.070 g di Likopen. Batasan inilebih rendah dari mereka baru saja melaporkan [39].Mengenai tokoferol, LODs berkisar antara 0.007 g untuk-tokoferol dan 0.067 g untuk - tokoferol, sementara mereka berkisar dari 0.004 g (untuk pheophytin b) untuk 0.080 g (untuk chlorophyllsklorofil b).Tergantung pada senyawa, LOQs berkisar dari 0,002 g untuk0.268 g (untuk phytofluene dan klorofil b ini, masing-masing). Thehasil yang diperoleh untuk batas kuantisasi menunjukkan bahwa usulan regu-metode ini cukup sensitif untuk penentuan Diet isoprenoid senyawa menarik.3.2. presisi dan akurasiPengulangan dan reproduktifitas dievaluasi dengan mempertimbangkan deviasi standar relatif (Tabel 2). Tentangpengulangan, nilai-nilai RSD metode untuk unsaponifiedcontoh di bawah adalah 7,2%. Nilai tertinggi berpadanan dengan - karoten (7.18%) dan terendah yang violaxanthin (0,58%). Keduanyanilai-nilai yang diperoleh dalam analisis wortel. RSD tertinggidiamati di reproduktibilitas berpadanan dengan - tokoferol disampel wortel (11.87%) dan terendah yang lutein (4.66%)dalam contoh chard. Meskipun demikian, sebagian besar nilai RSD Diperolehdi bawah 12%, yang dikonfirmasi reproduktibilitas tinggi darimetode